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sede ex-Scienze Biomediche Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa

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2022 - Molecular Pathways of Breast Cancer in Systemic Sclerosis: Exploratory Immunohistochemical Analysis from the Sclero-Breast Study [Articolo su rivista]
Isca, C.; Spinella, A.; Toss, A.; de Pinto, M.; Ficarra, G.; Fabbiani, L.; Iannone, A.; Magnani, L.; Lumetti, F.; Macripo, P.; Vacchi, C.; Gasparini, E.; Piana, S.; Cortesi, L.; Maiorana, A.; Salvarani, C.; Dominici, M.; Giuggioli, D.

Several authors reported an increased risk of cancer in SSc patients, including breast cancer (BC). Nevertheless, the mechanisms underlying this association have not yet been clarified. SSc and BC share several molecular pathways, which seem to play a common etiopathogenetic role. The previously published Sclero-Breast study demonstrated the development of BC with a good prognosis among these patients, which could be explained by an autoimmune background as a possible mechanism for limiting tumor extension. Here, we report the results of an IHC analysis of molecular pathways known to be common drivers for both diseases, with the aim to better define the mechanisms underlying a good prognosis of BC in patients affected by SSc. The analysis demonstrated higher TILs rates in all BC subgroups, with a high rate of PD-L1 expression especially in TNBC and HER2-positive BC, suggesting a less aggressive behavior in these patients compared to the general population. These results support a possible de-escalation strategy of cancer therapies in these fragile patients. These data could represent a starting point for future prospective studies based on the clinical application of these biomarkers with a larger sample size to promote a personalized and targeted oncological treatment for this specific subset of patients.

2021 - Author Correction: Endogenous control of inflammation characterizes pregnant women with asymptomatic or paucisymptomatic SARS-CoV-2 infection (Nature Communications, (2021), 12, 1, (4677), 10.1038/s41467-021-24940-w) [Articolo su rivista]
De Biasi, S.; Tartaro, D. L.; Gibellini, L.; Paolini, A.; Quong, A.; Petes, C.; Awong, G.; Douglas, S.; Lin, D.; Nieto, J.; Galassi, F. M.; Borella, R.; Fidanza, L.; Mattioli, M.; Leone, C.; Neri, I.; Meschiari, M.; Cicchetti, L.; Iannone, A.; Trenti, T.; Sarti, M.; Girardis, M.; Guaraldi, G.; Mussini, C.; Facchinetti, F.; Cossarizza, A.

The original version of this Article contained an error in Table 1. The correct version of the first row of the 2nd, 3rd, 5th and 7th columns states ‘CTR’, ‘PN’, ‘CTR vs PN’ and ‘PN vs PP’, instead of the original, incorrect ‘HD’, ‘NP’, ‘CTR vs NP’ and ‘CTR vs PP’. This has been corrected in both the PDF and HTML versions of the Article.

Toss, Angela; Spinella, Amelia; Isca, Chrystel; Vacchi, Caterina; Ficarra, Guido; Fabbiani, Luca; Iannone, Anna; Magnani, Luca; Castrignanò, Paola; Macripo', Pierluca; Gasparini, Elisa; Piana, Simonetta; Cortesi, Laura; Maiorana, Antonino; Salvarani, Carlo; Dominici, Massimo; Giuggioli, Dilia

Systemic Sclerosis (SSc) is a chronic disease associated with a 1.5-fold increase in cancer risk, including lung cancer, hematological malignancies, and breast cancer (BC). This is a retrospective study aiming to explore the clinical and pathological features of BC developed by SSc patients. A total of 54.5% of patients developed BC before SSc (median interval: 5 years), whereas 45.5% of patients developed BC after SSc (median delay: 8 years). A total of 93.1% of patients were diagnosed with an early stage tumor. Among invasive carcinomas, 70.8% presented with a low Mib1, 8.3% with a tubular histotype, and 42.8% with a Luminal A-like phenotype. A total of 66.6% of patients underwent breast-conserving surgery and 55.5% RT. A total of 40% of patients developed interstitial lung disease after RT and 20% diffuse cutaneous SSc. The cause of death of the six deceased patients was PAH. A significant association was observed between the use of immunosuppressive therapy and diffuse skin extension, negative ACA, positive Anti-Scl-70, and interstitial lung disease, but not BC status. SSc patients developed BC at a good prognosis, suggesting a de-escalation strategy of cancer therapies. In particular, ionizing radiation and chemotherapeuticals should be limited to higher-risk cases. Finally, proper screening is mandatory in order to allow for early cancer detection in SSc patients.

2021 - Clinical and Pathological Features of Breast Cancer in Systemic Sclerosis: Results from the Sclero-Breast Study [Articolo su rivista]
Toss, Angela; Spinella, Amelia; Isca, Chrystel; Vacchi, Caterina; Ficarra, Guido; Fabbiani, Luca; Iannone, Anna; Magnani, Luca; Castrignanò, Paola; Macripò, Pierluca; Gasparini, Elisa; Piana, Simonetta; Cortesi, Laura; Maiorana, Antonino; Salvarani, Carlo; Dominici, Massimo; Giuggioli, Dilia

Systemic Sclerosis (SSc) is a chronic disease associated with a 1.5-fold increase in cancer risk, including lung cancer, hematological malignancies, and breast cancer (BC). This is a retrospective study aiming to explore the clinical and pathological features of BC developed by SSc patients. A total of 54.5% of patients developed BC before SSc (median interval: 5 years), whereas 45.5% of patients developed BC after SSc (median delay: 8 years). A total of 93.1% of patients were diagnosed with an early stage tumor. Among invasive carcinomas, 70.8% presented with a low Mib1, 8.3% with a tubular histotype, and 42.8% with a Luminal A-like phenotype. A total of 66.6% of patients underwent breast-conserving surgery and 55.5% RT. A total of 40% of patients developed interstitial lung disease after RT and 20% diffuse cutaneous SSc. The cause of death of the six deceased patients was PAH. A significant association was observed between the use of immunosuppressive therapy and diffuse skin extension, negative ACA, positive Anti-Scl-70, and interstitial lung disease, but not BC status. SSc patients developed BC at a good prognosis, suggesting a de-escalation strategy of cancer therapies. In particular, ionizing radiation and chemotherapeuticals should be limited to higher-risk cases. Finally, proper screening is mandatory in order to allow for early cancer detection in SSc patients.

2021 - Endogenous control of inflammation characterizes pregnant women with asymptomatic or paucisymptomatic SARS-CoV-2 infection [Articolo su rivista]
De Biasi, S.; Tartaro, D. L.; Gibellini, L.; Paolini, A.; Quong, A.; Petes, C.; Awong, G.; Douglas, S.; Lin, D.; Nieto, J.; Galassi, F. M.; Borella, R.; Fidanza, L.; Mattioli, M.; Leone, C.; Neri, I.; Meschiari, M.; Cicchetti, L.; Iannone, A.; Trenti, T.; Sarti, M.; Girardis, M.; Guaraldi, G.; Mussini, C.; Facchinetti, F.; Cossarizza, A.

SARS-CoV-2 infection can affect all human beings, including pregnant women. Thus, understanding the immunological changes induced by the virus during pregnancy is nowadays of pivotal importance. Here, using peripheral blood from 14 pregnant women with asymptomatic or mild SARS-CoV-2 infection, we investigate cell proliferation and cytokine production, measure plasma levels of 62 cytokines, and perform a 38-parameter mass cytometry analysis. Our results show an increase in low density neutrophils but no lymphopenia or gross alterations of white blood cells, which display normal levels of differentiation, activation or exhaustion markers and show well preserved functionality. Meanwhile, the plasma levels of anti-inflammatory cytokines such as interleukin (IL)-1RA, IL-10 and IL-19 are increased, those of IL-17, PD-L1 and D-dimer are decreased, but IL-6 and other inflammatory molecules remain unchanged. Our profiling of antiviral immune responses may thus help develop therapeutic strategies to avoid virus-induced damages during pregnancy.

2021 - Hypokalemia in Patients with COVID-19 [Articolo su rivista]
Alfano, G.; Ferrari, A.; Fontana, F.; Perrone, R.; Mori, G.; Ascione, E.; Magistroni, R.; Venturi, G.; Pederzoli, S.; Margiotta, G.; Romeo, M.; Piccinini, F.; Franceschi, G.; Volpi, S.; Faltoni, M.; Ciusa, G.; Bacca, E.; Tutone, M.; Raimondi, A.; Menozzi, M.; Franceschini, E.; Cuomo, G.; Orlando, G.; Santoro, A.; Di Gaetano, M.; Puzzolante, C.; Carli, F.; Bedini, A.; Milic, J.; Meschiari, M.; Mussini, C.; Cappelli, G.; Guaraldi, G.; Borghi, V.; Burastero, G.; Corradi, L.; Di Gaetano, M.; Dolci, G.; Fantini, R.; Iadisernia, V.; Larne, D.; Pellegrino, F.; Rogati, C.; Santoro, A.; Tonelli, R.; Yaacoub, D.; Alfan, S.; Marco, B.; Pulizzi, R.; Leonelli, M.; Facchini, F.; Damiano, F.; Girardis, M.; Andreotti, A.; Biagioni, E.; Bondi, F.; Busani, S.; Chierego, G.; Scotti, M.; Cossarizza, L. S. A.; Bellinazzi, C.; Borella, R.; De Biasi, S.; De Gaetano, A.; Fidanza, L.; Gibellini, L.; Iannone, A.; Tartaro, D. L.; Mattioli, M.; Nasi, M.; Paolini, A.; Pinti, M.

Background: Patients with COVID-19 experience multiple clinical conditions that may cause electrolyte imbalances. Hypokalemia is a concerning electrolyte disorder closely associated with severe complications. This study aimed to estimate prevalence, risk factors and outcome of hypokalemia in a cohort of patients with confirmed COVID-19. Methods: A retrospective analysis was conducted on 290 non-ICU admitted patients with COVID-19 at the tertiary teaching hospital of Modena, Italy, from February 16 to April 14, 2020. Results: Hypokalemia was detected in 119 out of 290 patients (41%) during hospitalization. Mean serum potassium was 3.1 ± 0.1 meq/L. The majority of patients (90.7%) patients experienced only a mild decrease in serum potassium level (3–3.4 mEq/L). Hypokalemia was associated with hypocalcemia, which was detected in 50% of subjects. Urine potassium-to-creatinine ratio, measured in a small number of patients (n = 45; 36.1%), revealed an increase of urinary potassium excretion in most cases (95.5%). Risk factors for hypokalemia were female sex (odds ratio (OR) 2.44; 95% CI 1.36–4.37; P 0.003) and diuretic therapy (OR 1.94, 95% CI 1.08–3.48; P 0.027). Hypokalemia, adjusted for sex, age and SOFA score, was not associated with ICU transfer (OR 0.52; 95% CI 0.228–1.212; P = 0.131), in-hospital mortality (OR, 0.47; 95% CI 0.170–1.324; P = 0.154) and composite outcome of ICU transfer or in-hospital mortality (OR 0.48; 95% CI 0.222–1.047; P = 0.065) in our cohort of patients. Conclusions: Hypokalemia was a frequent disorder in subjects with COVID-19. Female sex and diuretic therapy were identified as risk factors for low serum potassium levels. Hypokalemia was unrelated to ICU transfer and death in this cohort of patients.

2021 - Obesity in Postmenopausal Breast Cancer Patients: It Is Time to Improve Actions for a Healthier Lifestyle. The Results of a Comparison Between Two Italian Regions With Different “Presumed” Lifestyles [Articolo su rivista]
Cortesi, L.; Galli, G. R.; Domati, F.; Conte, L.; Manca, L.; Berio, M. A.; Toss, A.; Iannone, A.; Federico, M.

Background: Adult body fatness is a convincing risk factor for postmenopausal breast cancer. With the aim to compare the different breast cancer (BC) features in Northern and Southern Italy, we investigated the relationship between BMI and BC characteristic in two groups of patients referred in the Modena and Lecce breast units. Materials and Methods: A retrospective analysis of a continuous series of BC patients referred to the Città di Lecce Hospital and the Modena Cancer Center, from January 2019 to December 2020 was performed. We identified four groups of BMI at BC diagnosis: underweight, BMI <18.5 kg/m2; normal weight, BMI ≥ 18.5–24.9 kg/m2; overweight, BMI ≥ 25.0–29.9 kg/m2; obese, BMI ≥30.0 kg/m2. BC characteristics and clinical outcomes were analyzed by the Kolmogorov-Smirnov test and Mann-Whitney U test; categorical data were compared using Pearson’s chi-square test, and dicotomic data were compared by odds ratio. Results: Nine hundred seventy-seven BC patients were included in the analysis. Overall, 470 were from Modena and 507 from Lecce. No differences were observed in the mean age of BC patients of Modena (61,42) and Lecce (62,08). No statistical differences between the two populations were shown in terms of tumor characteristics and pathological stage. Conversely, a statistical difference of BMI between the BC patients coming from Modena and Lecce (25.87 and 27.81, respectively; p = 0.000001) was found. BC patients diagnosed in Lecce at age ≥70 years had higher median BMI compared with the ones from Modena (p = 0.000002). The increased BMI in this aged population was also associated to larger tumor size (p = 0.040). Conclusion: The rate of overweight and obesity was higher in BC women living in Southern Italy, despite the presumed nutrition according to the so-called Mediterranean type dietary pattern. Unexpectedly, an increased BMI rate and a relationship with larger tumor size were found in Southern BC patients aged ≥70 years. Our findings strongly support the need for promoting a healthier lifestyle model in Italy, with the aim of reducing the rate of the obesity and, consequently, the increased risk of BC.

2020 - Increased plasma levels of mitochondrial DNA and pro-inflammatory cytokines in patients with progressive multiple sclerosis [Articolo su rivista]
Nasi, M.; Bianchini, E.; De Biasi, S.; Gibellini, L.; Neroni, A.; Mattioli, Marco; Pinti, M.; Iannone, A.; Mattioli, A. V.; Simone, A. M.; Ferraro, D.; Vitetta, F.; Sola, P.; Cossarizza, A.

The role of damage-associated molecular patterns in multiple sclerosis (MS) is under investigation. Here, we studied the contribution of circulating high mobility group box protein 1 (HMGB1) and mitochondrial DNA (mtDNA) to neuroinflammation in progressive MS. We measured plasmatic mtDNA, HMGB1 and pro-inflammatory cytokines in 38 secondary progressive (SP) patients, 35 primary progressive (PP) patients and 42 controls. Free mtDNA was higher in SP than PP. Pro-inflammatory cytokines were increased in progressive patients. In PP, tumor necrosis factor-α correlated with MS Severity Score. Thus, in progressive patients, plasmatic mtDNA and pro-inflammatory cytokines likely contribute to the systemic inflammatory status.

2020 - Lifestyle intervention on body weight and physical activity in patients with breast cancer can reduce the risk of death in obese women: The EMILI study [Articolo su rivista]
Cortesi, L.; Sebastiani, F.; Iannone, A.; Marcheselli, L.; Venturelli, M.; Piombino, C.; Toss, A.; Federico, M.

Background obesity and sedentary lifestyle have been shown to negatively affect survival in breast cancer (BC). The purpose of this study was to test the efficacy of a lifestyle intervention on body mass index (BMI) and physical activity (PA) levels among BC survivors in Modena, Italy, in order to show an outcome improvement in obese and overweight patients. Methods: This study is a single-arm experimental design, conducted between November 2009 and May 2016 on 430 women affected by BC. Weight, BMI, and PA were assessed at baseline, at 12 months, and at the end of the study. Survival curves were estimated among normal, overweight, and obese patients. Results: Mean BMI decreased from baseline to the end of the study was equal to 2.9% (p = 0.065) in overweight patients and 3.3% in obese patients (p = 0.048). Mean PA increase from baseline to the end of the study was equal to 125% (p < 0.001) in normal patients, 200% (p < 0.001) in overweight patients and 100% (p < 0.001) in obese patients. After 70 months of follow-up, the 5-year overall survival (OS) rate was 96%, 96%, and 93%, respectively in normal, obese, and overweight patients. Overweight patients had significantly worse OS than normal ones (HR = 3.69, 95%CI = 1.82–4.53 p = 0.027) whereas no statistically significant differences were seen between obese and normal patients (HR 2.45, 95%CI = 0.68–8.78, p = 0.169). Conclusions: A lifestyle intervention can lead to clinically meaningful weight loss and increase PA in patients with BC. These results could contribute to improving the OS in obese patients compared to overweight ones.

2020 - Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with Covid-19 pneumonia. [Articolo su rivista]
De Biasi, S; Meschiari, M; Gibellini, L; Bellinazzi, C; Borella, R; Fidanza, L; Gozzi, L; Iannone, A; Lo Tartaro, D; Mattioli, M; Paolini, A; Menozzi, M; Milić, J; Franceschi, G; Fantini, R; Tonelli, R; Sita, M; Sarti, M; Trenti, T; Brugioni, L; Cicchetti, L; Facchinetti, F; Pietrangelo, A; Clini, E; Girardis, M; Guaraldi, G; Mussini, C; Cossarizza, A.

We provide an in-depth investigation of the T cell compartment and functionality, cytokine production and plasma levels in a total of 39 patients affected by Covid-19 pneumonia. At admission, patients were lymphopenic; for all, SARS-CoV-2 was detected in a nasopharyngeal swab specimen by real-time RT-PCR, and pneumonia was subsequently confirmed by X-rays. Detailed 18-parameter flow cytometry coupled with unsupervised data analysis revealed that patients showed similar percentages of CD4+ and CD8+ T cells, but a decreased absolute number in both populations. For CD4+ T lymphocytes, we found a significant decrease in the number of naïve, central and effector memory cells and an increased percentage of terminally differentiated cells, regulatory T cells, and of those that were activated or that were expressing PD1 and CD57 markers. Studies on chemokine receptors and lineage-specifying transcription factors revealed that, among CD4+ T cells, patients displayed a lower percentage of cells expressing CCR6 or CXCR3, and of those co-expressing CCR6 and CD161, but higher percentages of 62 CXCR4+ or CCR4+ cells. No differences were noted in the expression of T-bet or GATA-3. Analyses of patients' CD8+ T cells showed decreased numbers of naïve and central memory and increased amounts of activated cells, accompanied by increased percentages of activated cells and of lymphocytes expressing CD57, PD1, or both. CD8+ T cells expressed lower percentages of CCR6+, CXCR3+ or T-bet+ cells and of CXCR3+,T-bet+ or CCR6+,CD161+ lymphocytes. We also found higher percentages of cells expressing CCR4+, CXCR4 or GATA-3. Analyses of lymphocyte proliferation revealed that terminally differentiated CD4+ and CD8+ T cell from patients had a lower proliferative index than controls, whereas cellular bioenergetics, measured by the quantification of mitochondrial oxygen consumption and extracellular acidification rate, was similar in CD4+ T cells from both groups. We measured plasma level of 31 cytokines linked to inflammation, including T helper (TH)type-1 and TH2 cytokines, chemokines, galectins, pro- and anti-inflammatory mediators, finding that most were dramatically increased in Covid-19 patients, confirming the presence of a massive cytokine storm. Analysis of the production of different cytokines after stimulation by anti-CD3/CD28 monoclonal antibodies revealed that patients not only had a high capacity to produce tumour necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-2, but also showed a significant skewing of CD4+ T cells towards the TH17 phenotype. A therapeutic approach now exists based on the administration of drugs that block IL-6pathway, and seems to improve the disease. IL-17 is crucial in recruiting and activating neutrophils, cells that can migrate to the lung and are heavily involved in the pathogenesis of Covid-19. We show here that a skewing of activated T cells towards the TH17 functional phenotype exists in Covid-19 patients. We therefore suggest that blocking the IL-17 pathway by biological drugs that are already used to treat different pathologies could provide a novel, additional strategy to improve the health of patients infected by SARS-CoV-2.

2020 - Peritoneal dialysis in the time of coronavirus disease 2019 [Articolo su rivista]
Alfano, Gaetano; Fontana, Francesco; Ferrari, Annachiara; Guaraldi, Giovanni; Mussini, Cristina; Magistroni, Riccardo; Cappelli, Gianni; Bacca, Erica; Bedini, Andrea; Borghi, Vanni; Burastero, Giulia; Carli, Federica; Ciusa, Giacomo; Corradi, Luca; Cuomo, Gianluca; Digaetano, Margherita; Dolci, Giovanni; Faltoni, Matteo; Fantini, Riccardo; Franceschi, Giacomo; Franceschini, Ericad; Iadisernia, Vittorio; Larnõ, Damiano; Menozzi, Marianna; Meschiari, Marianna; Milic, Jovana; Orlando, Gabriella; Pellegrino, Francesco; Raimondi, Alessandro; Rogati, Carlotta; Santoro, Antonella; Tonelli, Roberto; Tutone, Marco; Volpi, Sara; Yaacoub, Dina; Aten, G.; Marco, Ballestri; Mori, Giacomo; Girardis, Massimo; Andreotti, Alberto; Biagioni, Emanuela; Bondi, Filippo; Busani, Stefano; Chierego, Giovanni; Scotti, Marzia; Serio, Lucia; Cossarizza, Andrea; Bellinazzi, Caterina; Borella, Rebecca; de Biasi, Sara; de Gaetano, Anna; Fidanza, Lucia; Gibellini, Lara; Iannone, Anna; Lo Tartaro, Domenico; Mattioli, Marco; Nasi, Milena; Paolini, Annamariag; Pinti, Marcello

In the current setting of global containment, peritoneal dialysis (PD) and home haemodialysis are the best modalities of renal replacement therapy (RRT) to reduce the rate of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Considering the shorter and easier training programme of PD compared to home haemodialysis, PD appears a practical solution for patients with end-stage renal disease to reduce the risk of hospital-acquired infection. PD offers the advantage of minimizing the risk of viral transmission through interpersonal contact that commonly occurs during the haemodialysis session and while travelling from home to the haemodialysis facility using public transport services. To overcome barriers to health care access due to the containment measures for this emerging disease, telemedicine is a useful and reliable tool for delivering health care without exposing patients to the risk of contact. However, novel issues including handling of potentially infected dialysate, caregivers' infectious risk and adequacy of PD in critically ill patients with acute respiratory distress syndrome remain to be clarified. In conclusion, PD should be preferred to the other modalities of RRT during the coronavirus disease 2019 (COVID-19) outbreak because it can be a solution to cope with the increased number of infected patients worldwide.

2019 - Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition) [Articolo su rivista]
Cossarizza, A.; Chang, H. -D.; Radbruch, A.; Acs, A.; Adam, D.; Adam-Klages, S.; Agace, W. W.; Aghaeepour, N.; Akdis, M.; Allez, M.; Almeida, L. N.; Alvisi, G.; Anderson, G.; Andra, I.; Annunziato, F.; Anselmo, A.; Bacher, P.; Baldari, C. T.; Bari, S.; Barnaba, V.; Barros-Martins, J.; Battistini, L.; Bauer, W.; Baumgart, S.; Baumgarth, N.; Baumjohann, D.; Baying, B.; Bebawy, M.; Becher, B.; Beisker, W.; Benes, V.; Beyaert, R.; Blanco, A.; Boardman, D. A.; Bogdan, C.; Borger, J. G.; Borsellino, G.; Boulais, P. E.; Bradford, J. A.; Brenner, D.; Brinkman, R. R.; Brooks, A. E. S.; Busch, D. H.; Buscher, M.; Bushnell, T. P.; Calzetti, F.; Cameron, G.; Cammarata, I.; Cao, X.; Cardell, S. L.; Casola, S.; Cassatella, M. A.; Cavani, A.; Celada, A.; Chatenoud, L.; Chattopadhyay, P. K.; Chow, S.; Christakou, E.; Cicin-Sain, L.; Clerici, M.; Colombo, F. S.; Cook, L.; Cooke, A.; Cooper, A. M.; Corbett, A. J.; Cosma, A.; Cosmi, L.; Coulie, P. G.; Cumano, A.; Cvetkovic, L.; Dang, V. D.; Dang-Heine, C.; Davey, M. S.; Davies, D.; De Biasi, S.; Del Zotto, G.; Dela Cruz, G. V.; Delacher, M.; Della Bella, S.; Dellabona, P.; Deniz, G.; Dessing, M.; Di Santo, J. P.; Diefenbach, A.; Dieli, F.; Dolf, A.; Dorner, T.; Dress, R. J.; Dudziak, D.; Dustin, M.; Dutertre, C. -A.; Ebner, F.; Eckle, S. B. G.; Edinger, M.; Eede, P.; Ehrhardt, G. R. A.; Eich, M.; Engel, P.; Engelhardt, B.; Erdei, A.; Esser, C.; Everts, B.; Evrard, M.; Falk, C. S.; Fehniger, T. A.; Felipo-Benavent, M.; Ferry, H.; Feuerer, M.; Filby, A.; Filkor, K.; Fillatreau, S.; Follo, M.; Forster, I.; Foster, J.; Foulds, G. A.; Frehse, B.; Frenette, P. S.; Frischbutter, S.; Fritzsche, W.; Galbraith, D. W.; Gangaev, A.; Garbi, N.; Gaudilliere, B.; Gazzinelli, R. T.; Geginat, J.; Gerner, W.; Gherardin, N. A.; Ghoreschi, K.; Gibellini, L.; Ginhoux, F.; Goda, K.; Godfrey, D. I.; Goettlinger, C.; Gonzalez-Navajas, J. M.; Goodyear, C. S.; Gori, A.; Grogan, J. L.; Grummitt, D.; Grutzkau, A.; Haftmann, C.; Hahn, J.; Hammad, H.; Hammerling, G.; Hansmann, L.; Hansson, G.; Harpur, C. M.; Hartmann, S.; Hauser, A.; Hauser, A. E.; Haviland, D. L.; Hedley, D.; Hernandez, D. C.; Herrera, G.; Herrmann, M.; Hess, C.; Hofer, T.; Hoffmann, P.; Hogquist, K.; Holland, T.; Hollt, T.; Holmdahl, R.; Hombrink, P.; Houston, J. P.; Hoyer, B. F.; Huang, B.; Huang, F. -P.; Huber, J. E.; Huehn, J.; Hundemer, M.; Hunter, C. A.; Hwang, W. Y. K.; Iannone, A.; Ingelfinger, F.; Ivison, S. M.; Jack, H. -M.; Jani, P. K.; Javega, B.; Jonjic, S.; Kaiser, T.; Kalina, T.; Kamradt, T.; Kaufmann, S. H. E.; Keller, B.; Ketelaars, S. L. C.; Khalilnezhad, A.; Khan, S.; Kisielow, J.; Klenerman, P.; Knopf, J.; Koay, H. -F.; Kobow, K.; Kolls, J. K.; Kong, W. T.; Kopf, M.; Korn, T.; Kriegsmann, K.; Kristyanto, H.; Kroneis, T.; Krueger, A.; Kuhne, J.; Kukat, C.; Kunkel, D.; Kunze-Schumacher, H.; Kurosaki, T.; Kurts, C.; Kvistborg, P.; Kwok, I.; Landry, J.; Lantz, O.; Lanuti, P.; Larosa, F.; Lehuen, A.; LeibundGut-Landmann, S.; Leipold, M. D.; Leung, L. Y. T.; Levings, M. K.; Lino, A. C.; Liotta, F.; Litwin, V.; Liu, Y.; Ljunggren, H. -G.; Lohoff, M.; Lombardi, G.; Lopez, L.; Lopez-Botet, M.; Lovett-Racke, A. E.; Lubberts, E.; Luche, H.; Ludewig, B.; Lugli, E.; Lunemann, S.; Maecker, H. T.; Maggi, L.; Maguire, O.; Mair, F.; Mair, K. H.; Mantovani, A.; Manz, R. A.; Marshall, A. J.; Martinez-Romero, A.; Martrus, G.; Marventano, I.; Maslinski, W.; Matarese, G.; Mattioli, A. V.; Maueroder, C.; Mazzoni, A.; Mccluskey, J.; Mcgrath, M.; Mcguire, H. M.; Mcinnes, I. B.; Mei, H. E.; Melchers, F.; Melzer, S.; Mielenz, D.; Miller, S. D.; Mills, K. H. G.; Minderman, H.; Mjosberg, J.; Moore, J.; Moran, B.; Moretta, L.; Mosmann, T. R.; Muller, S.; Multhoff, G.; Munoz, L. E.; Munz, C.; Nakayama, T.; Nasi, M.; Neumann, K.; Ng, L. G.; Niedobitek, A.; Nourshargh, S.; Nunez, G.; O'Connor, J. -E.; Ochel, A.; Oja, A.; Ordonez, D.; Orfao, A.; Orlowski-Oliver, E.; Ouyang, W.; Oxenius, A.; Palankar, R.; Panse, I.; Pattanapanyasat, K.; Paulsen, M.; Pavlinic, D.; Penter,

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

2018 - Exploring viral reservoir: The combining approach of cell sorting and droplet digital PCR [Articolo su rivista]
Gibellini, Lara; Pecorini, Simone; De Biasi, Sara; Pinti, Marcello; Bianchini, Elena; DE GAETANO, Anna; Digaetano, Margherita; Pullano, Rosalberta; Lo Tartaro, Domenico; Iannone, Anna; Mussini, Cristina; Cossarizza, Andrea; Nasi, Milena

Combined antiretroviral therapy (cART) blocks different steps of HIV replication and maintains plasma viral RNA at undetectable levels. The virus can remain in long-living cells and create a reservoir where HIV can restart replicating after cART discontinuation. A persistent viral production triggers and maintains a persistent immune activation, which is a well-known feature of chronic HIV infection, and contributes either to precocious aging, or to the increased incidence of morbidity and mortality of HIV positive patients. The new frontier of the treatment of HIV infection is nowadays eradication of the virus from all host cells and tissues. For this reason, it is crucial to have a clear and precise idea of where the virus hides, and which are the cells that keep it silent. Important efforts have been made to improve the detection of viral reservoirs, and new techniques are now giving the opportunity to characterize viral reservoirs. Among these techniques, a strategic approach based upon cell sorting and droplet digital PCR (ddPCR) is opening new horizons and opportunities of research. This review provides an overview of the methods that combine cell sorting and ddPCR for the quantification of HIV DNA in different cell types, and for the detection of its maintenance.

2018 - Genomic alterations at the basis of treatment resistance in metastatic breast cancer: Clinical applications [Articolo su rivista]
Toss, Angela; Piacentini, Federico; Cortesi, Laura; Artuso, Lucia; Bernardis, Isabella; Parenti, Sandra; Tenedini, Elena; Ficarra, Guido; Maiorana, Antonino; Iannone, Anna; Omarini, Claudia; Moscetti, Luca; Cristofanilli, Massimo; Federico, Massimo; Tagliafico, Enrico

The standard of care for breast cancer has gradually evolved from empirical treatments based on clinical-pathological characteristics to the use of targeted approaches based on the molecular profile of the tumor. Consequently, an increasing number of molecularly targeted drugs have been developed. These drugs target specific alterations, called driver mutations, which confer a survival advantage to cancer cells. To date, the main challenge remains the identification of predictive biomarkers for the selection of the optimal treatment. On this basis, we evaluated a panel of 25 genes involved in the mechanisms of targeted treatment resistance, in 16 primary breast cancers and their matched recurrences, developed during treatment. Overall, we found a detection rate of mutations higher than that described in the literature. In particular, the most frequently mutated genes were ERBB2 and those involved in the PI3K/AKT/mTOR and the MAPK signaling pathways. The study revealed substantial discordances between primary tumors and metastases, stressing the need for analysis of metastatic tissues at recurrence. We observed that 85.7% of patients with an early-stage or locally advanced primary tumor showed at least one mutation in the primary tumor. This finding could explain the subsequent relapse and might therefore justify more targeted adjuvant treatments. Finally, the mutations detected in 50% of relapsed tissues could have guided subsequent treatment choices in a different way. This study demonstrates that mutation events may be present at diagnosis or arise during cancer treatment. As a result, profiling primary and metastatic tumor tissues may be a major step in defining optimal treatments.

2018 - LonP1 Differently Modulates Mitochondrial Function and Bioenergetics of Primary Versus Metastatic Colon Cancer Cells [Articolo su rivista]
Gibellini, L; Losi, L; De Biasi, S; Nasi, M; Lo Tartaro, D; Pecorini, S; Patergnani, S; Pinton, P; De Gaetano, A; Carnevale, G; Pisciotta, A; Mariani, F; Roncucci, L; Iannone, A; Cossarizza, A; Pinti, M.

Mitochondrial Lon protease (LonP1) is a multi-function enzyme that regulates mitochondrial functions in several human malignancies, including colorectal cancer (CRC). The mechanism(s) by which LonP1 contributes to colorectal carcinogenesis is not fully understood. We found that silencing LonP1 leads to severe mitochondrial impairment and apoptosis in colon cancer cells. Here, we investigate the role of LonP1 in mitochondrial functions, metabolism, and epithelial-mesenchymal transition (EMT) in colon tumor cells and in metastasis. LonP1 was almost absent in normal mucosa, gradually increased from aberrant crypt foci to adenoma, and was most abundant in CRC. Moreover, LonP1 was preferentially upregulated in colorectal samples with mutated p53 or nuclear β-catenin, and its overexpression led to increased levels of β-catenin and decreased levels of E-cadherin, key proteins in EMT, in vitro. LonP1 upregulation also induced opposite changes in oxidative phosphorylation, glycolysis, and pentose pathway in SW480 primary colon tumor cells when compared to SW620 metastatic colon cancer cells. In conclusion, basal LonP1 expression is essential for normal mitochondrial function, and increased LonP1 levels in SW480 and SW620 cells induce a metabolic shift toward glycolysis, leading to EMT.

2017 - Guidelines for the use of flow cytometry and cell sorting in immunological studies [Articolo su rivista]
Cossarizza, Andrea; Chang, Hyun-dong; Radbruch, Andreas; Andrã¤, Immanuel; Annunziato, Francesco; Bacher, Petra; Barnaba, Vincenzo; Battistini, Luca; Bauer, Wolfgang M.; Baumgart, Sabine; Becher, Burkhard; Beisker, Wolfgang; Berek, Claudia; Blanco, Alfonso; Borsellino, Giovanna; Boulais, Philip E.; Brinkman, Ryan R.; Bã¼scher, Martin; Busch, Dirk H.; Bushnell, Timothy P.; Cao, Xuetao; Cavani, Andrea; Chattopadhyay, Pratip K.; Cheng, Qingyu; Chow, Sue; Clerici, Mario; Cooke, Anne; Cosma, Antonio; Cosmi, Lorenzo; Cumano, Ana; Dang, Van Duc; Davies, Derek; De Biasi, Sara; Del Zotto, Genny; Della Bella, Silvia; Dellabona, Paolo; Deniz, Gã¼nnur; Dessing, Mark; Diefenbach, Andreas; Di Santo, James; Dieli, Francesco; Dolf, Andreas; Donnenberg, Vera S.; Dã¶rner, Thomas; Ehrhardt, Gã¶tz R. A.; Endl, Elmar; Engel, Pablo; Engelhardt, Britta; Esser, Charlotte; Everts, Bart; Falk, Christine S.; Fehniger, Todd A.; Filby, Andrew; Fillatreau, Simon; Follo, Marie; Fã¶rster, Irmgard; Foster, John; Foulds, Gemma A.; Frenette, Paul S.; Galbraith, David; Garbi, Natalio; Garcã­a-godoy, Maria Dolores; Ghoreschi, Kamran; Gibellini, Lara; Goettlinger, Christoph; Goodyear, Carl S.; Gori, Andrea; Grogan, Jane; Gross, Mor; Grã¼tzkau, Andreas; Grummitt, Daryl; Hahn, Jonas; Hammer, Quirin; Hauser, Anja E.; Haviland, David L.; Hedley, David; Herrera, Guadalupe; Herrmann, Martin; Hiepe, Falk; Holland, Tristan; Hombrink, Pleun; Houston, Jessica P.; Hoyer, Bimba F.; Huang, Bo; Hunter, Christopher A.; Iannone, Anna; Jã¤ck, Hans-martin; Jã¡vega, Beatriz; Jonjic, Stipan; Juelke, Kerstin; Jung, Steffen; Kaiser, Toralf; Kalina, Tomas; Keller, Baerbel; Khan, Srijit; Kienhã¶fer, Deborah; Kroneis, Thomas; Kunkel, Dã©sirã©e; Kurts, Christian; Kvistborg, Pia; Lannigan, Joanne; Lantz, Olivier; Larbi, Anis; Leibundgut-landmann, Salome; Leipold, Michael D.; Levings, Megan K.; Litwin, Virginia; Liu, Yanling; Lohoff, Michael; Lombardi, Giovanna; Lopez, Lilly; Lovett-racke, Amy; Lubberts, Erik; Ludewig, Burkhard; Lugli, Enrico; Maecker, Holden T.; Martrus, Glã²ria; Matarese, Giuseppe; Mauerã¶der, Christian; Mcgrath, Mairi; Mcinnes, Iain; Mei, Henrik E.; Melchers, Fritz; Melzer, Susanne; Mielenz, Dirk; Mills, Kingston; Mjã¶sberg, Jenny; Moore, Jonni; Moran, Barry; Moretta, Alessandro; Moretta, Lorenzo; Mosmann, Tim R.; Mã¼ller, Susann; Mã¼ller, Werner; Mã¼nz, Christian; Multhoff, Gabriele; Munoz, Luis Enrique; Murphy, Kenneth M.; Nakayama, Toshinori; Nasi, Milena; Neudã¶rfl, Christine; Nolan, John; Nourshargh, Sussan; O'connor, Josã©-enrique; Ouyang, Wenjun; Oxenius, Annette; Palankar, Raghav; Panse, Isabel; Peterson, Pã¤rt; Peth, Christian; Petriz, Jordi; Philips, Daisy; Pickl, Winfried; Piconese, Silvia; Pinti, Marcello; Pockley, A. Graham; Podolska, Malgorzata Justyna; Pucillo, Carlo; Quataert, Sally A.; Radstake, Timothy R. D. J.; Rajwa, Bartek; Rebhahn, Jonathan A.; Recktenwald, Diether; Remmerswaal, Ester B. M.; Rezvani, Katy; Rico, Laura G.; Robinson, J. Paul; Romagnani, Chiara; Rubartelli, Anna; Ruland, Jã¼rgen; Sakaguchi, Shimon; Sala-de-oyanguren, Francisco; Samstag, Yvonne; Sanderson, Sharon; Sawitzki, Birgit; Scheffold, Alexander; Schiemann, Matthias; Schildberg, Frank; Schimisky, Esther; Schmid, Stephan A; Schmitt, Steffen; Schober, Kilian; Schã¼ler, Thomas; Schulz, Axel Ronald; Schumacher, Ton; Scotta, Cristiano; Shankey, T. Vincent; Shemer, Anat; Simon, Anna-katharina; Spidlen, Josef; Stall, Alan M.; Stark, Regina; Stehle, Christina; Stein, Merle; Steinmetz, Tobit; Stockinger, Hannes; Takahama, Yousuke; Tarnok, Attila; Tian, Zhigang; Toldi, Gergely; Tornack, Julia; Traggiai, Elisabetta; Trotter, Joe; Ulrich, Henning; Van Der Braber, Marlous; Van Lier, Renã© A. W.; Veldhoen, Marcello; Vento-asturias, Salvador; Vieira, Paulo; Voehringer, David; Volk, Hans-dieter; Von Volkmann, Konrad; Waisman, Ari; Walker, Rachael; Ward, Michael D.; Warnatz, Klaus; Warth, Sarah; Watson, James V.; Watzl, Carsten; Wegener, Leonie; Wi

Nessun abstract disponibile

2016 - Anti-TNF-α drugs differently affect the TNFa-sTNFR system and monocyte subsets in patients with psoriasis [Articolo su rivista]
Gibellini, Lara; De Biasi, Sara; Bianchini, Elena; Bartolomeo, Regina; Fabiano, Antonella; Manfredini, Marco; Ferrar, Federica; Albertini, Giuseppe; Trenti, Tommaso; Nasi, Milena; Pinti, Marcello; Iannone, Anna; Salvarani, Carlo; Cossarizza, Andrea; Pellacani, Giovanni

TNF-a has a central role in the development and maintenance of psoriatic plaques, and its serum levels correlate with disease activity. Anti-TNF-a drugs are, however, ineffective in a relevant percentage of patients for reasons that are currently unknown. To understand whether the response to anti-TNF-a drugs is influenced by the production of anti-drug antibodies or by the modulation of the TNFa-TNFa receptor system, and to identify changes in monocyte phenotype and activity, we analysed 119 psoriatic patients who either responded or did not respond to different anti-TNF-a therapies (adalimumab, etanercept or infliximab), and measured plasma levels of TNF-a, TNF-a soluble receptors, drug and anti-drug antibodies. Moreover, we analyzed the production of TNF-a and TNF-α soluble receptors by peripheral blood mononuclear cells (PBMCs), and characterized different monocyte populations. We found that: i) the drug levels varied between responders and non-responders; ii) anti-infliximab antibodies were present in 15% of infliximab-treated patients, while anti-etanercept or anti-adalimumab antibodies were never detected; iii) plasma TNF-a levels were higher in patients treated with etanercept compared to patients treated with adalimumab or infliximab; iv) PBMCs from patients responding to adalimumab and etanercept produced more TNF-a and sTNFRII in vitro than patients responding to infliximab; v) PBMCs from patients not responding to infliximab produce higher levels of TNF-a and sTNFRII than patients responding to infliximab; vi) anti- TNF-a drugs significantly altered monocyte subsets. A complex remodelling of the TNFa-TNFa receptor system thus takes place in patients treated with anti-TNF-α drugs, that involves either the production of anti-drug antibodies or the modulation of monocyte phenotype or inflammatory activity.

2016 - Effect of diets supplemented with different conjugated linoleic acid (CLA) isomers on protein expression in C57/BL6 mice [Articolo su rivista]
DELLA CASA, Lara; Rossi, Elena; Romanelli, Claudia; Gibellini, Lara; Iannone, Anna

The individual genetic variations, as a response to diet, have recently caught the attention of several researchers. In addition, there is also a trend to assume food containing beneficial substances, or to supplement food with specific compounds. Among these, there is the conjugated linoleic acid (CLA), which has been demonstrated to reduce fat mass and to increase lean mass, even though its mechanism of action is still not known. We investigated the effect of CLA isomers (CLA c9,t11 and CLA t10,c12) on the proteomic profile of liver, adipose tissue, and muscle of mouse, with the aim of verifying the presence of a modification in fat and lean mass, and to explore the mechanism of action.

2016 - Emerging role of Lon protease as a master regulator of mitochondrial functions [Articolo su rivista]
Pinti, Marcello; Gibellini, Lara; Nasi, Milena; De Biasi, Sara; Bortolotti, Carlo Augusto; Iannone, Anna; Cossarizza, Andrea

Lon protease is a nuclear-encoded, mitochondrial ATP-dependent protease highly conserved throughout the evolution, crucial for the maintenance of mitochondrial homeostasis. Lon acts as a chaperone of misfolded proteins, and is necessary for maintaining mitochondrial DNA. The impairment of these functions has a deep impact on mitochondrial functionality and morphology. An altered expression of Lon leads to a profound reprogramming of cell metabolism, with a switch from respiration to glycolysis, which is often observed in cancer cells. Mutations of Lon, which likely impair its chaperone properties, are at the basis of a genetic inherited disease named of the cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

2016 - Mitochondrial proteases as emerging pharmacological targets [Articolo su rivista]
Gibellini, Lara; DE BIASI, Sara; Nasi, Milena; Iannone, Anna; Cossarizza, Andrea; Pinti, Marcello

The preservation of mitochondrial function and integrity is critical for cell viability. Under stress conditions, unfolded, misfolded or damaged proteins accumulate in a certain compartment of the organelle, interfering with oxidative phosphorylation and normal mitochondrial functions. In stress conditions, several mechanisms, including mitochondrial unfolded protease response (UPRmt), fusion and fission, and mitophagy are engaged to restore normal proteostasis of the organelle. Mitochondrial proteases are a family of more than 20 enzymes that not only are involved in the UPRmt, but actively participate at multiple levels in the stress-response system. Alterations in their expression levels, or mutations that determine loss or gain of function of these proteases deeply impair mitochondrial functionality and can be associated with the onset of inherited diseases, with the development of neurodegenerative disorders and with the process of carcinogenesis. In this review, we focus our attention on six of them, namely CLPP, HTRA2 and LONP1, by analysing the current knowledge about their functions, their involvement in the pathogenesis of human diseases, and the compounds currently available for inhibiting their functions.

2013 - Ovarian cancer: can proteomics give new insights for therapy and diagnosis? [Articolo su rivista]
Toss, Angela; DE MATTEIS, Elisabetta; Rossi, Elena; Casa, L. D.; Iannone, Anna; Federico, Massimo; Cortesi, Laura

The study of the ovarian proteomic profile represents a new frontier in ovarian cancer research, since this approach is able to enlighten the wide variety of post-translational events (such as glycosylation and phosphorylation). Due to the possibility of analyzing thousands of proteins, which could be simultaneously altered, comparative proteomics represent a promising model of possible biomarker discovery for ovarian cancer detection and monitoring. Moreover, defining signaling pathways in ovarian cancer cells through proteomic analysis offers the opportunity to design novel drugs and to optimize the use of molecularly targeted agents against crucial and biologically active pathways. Proteomic techniques provide more information about different histological types of ovarian cancer, cell growth and progression, genes related to tumor microenvironment and specific molecular targets predictive of response to chemotherapy than sequencing or microarrays. Estimates of specificity with proteomics are less consistent, but suggest a new role for combinations of biomarkers in early ovarian cancer diagnosis, such as the OVA1 test. Finally, the definition of the proteomic profiles in ovarian cancer would be accurate and effective in identifying which pathways are differentially altered, defining the most effective therapeutic regimen and eventually improving health outcomes.

2012 - Conjugated linoleic acid alters lipid metabolism in hepatocytes [Abstract in Atti di Convegno]
Iannone, Anna; Rossi, Elena; DELLA CASA, Lara; Barchetti, Andrea; Battistini, Nino Carlo

not available

2012 - Conjugated linoleic acid isomers modulate protein expression profile in rat hepatocytes [Articolo su rivista]
Rossi, Elena; DELLA CASA, Lara; S., Piana; Iannone, Anna

Conjugated linoleic acid (CLA) is a polyunsaturatedfatty acid, which has been recently proven to beeffective in reducing body fat mass, but brings as a sideeffect, the liver enlargement due to an increased lipidcontent. The in vivo lipogenic activity has been suggestedto be due to the reduction in fat mass and to the consequentmetabolism of blood glucose to fatty acid in the liver ratherthan in the adipose tissue. We investigated the ability ofCLA to directly induce steatosis by modulating theexpression pattern of hepatic proteins involved in lipidmetabolism. To avoid interferences derived from CLAmetabolism by other tissues, we used the in vitro model offreshly isolated rat hepatocytes incubated in the presence ofdifferent CLA isomers. The direct effect of CLA on lipidaccumulation in hepatocytes was demonstrated by thealtered expression pattern of several proteins involved inlipid metabolism, as assessed by two-dimensional gelelectrophoresis and confirmed by Western blotting analysis.The CLA isomer c9,t11 was most effective in modulatingthe protein expression profile.

2012 - The impact of proteomics in the understanding of the molecular basis of paclitaxel-resistance in ovarian tumors [Articolo su rivista]
Vergara, D; Tinelli, A; Iannone, Anna; Maffia, M.

The current therapy for ovarian cancer has advanced from alkylating agents, to a combination of carboplatinum and paclitaxel offering increased survival. Although most patients respond to this first-line therapy, initially, the majority of these patients relapse within 2 years. The mechanisms responsible for acquired drug resistance in ovarian cancer have been elucidated only in part. They include i) enhanced drug export, ii) activation/inhibition of intracellular signalling pathways, iii) molecular alterations in tubulin isotype composition. A better understanding of these mechanisms is needed, in order to develop new approaches, aimed at overcoming resistance to anticancer agents, and to reveal the complexity of causes, which contribute to drug resistance. In this review we offer an updated overview of proteomic studies on the molecular mechanisms of paclitaxel resistance. These proteomic studies also identify potential targets for modulating drug resistance, that could be predictive of response to chemotherapy in ovarian carcinomas.

2011 - Protein expression patterns associated with advanced stage ovarian cancer [Articolo su rivista]
Cortesi, L.; Rossi, Elena; DELLA CASA, Lara; Barchetti, Andrea; Nicoli, A.; Piana, S.; Abrate, M.; LA SALA, Giovanni Battista; Federico, Massimo; Iannone, Anna

This is a comparative proteomic study on biopsies from patients with ovarian cancer to identify potential diagnostic/prognostic biomarkers in both healthy and tumor tissue, interstitial fluid (normal interstitial fluid and tumoral interstitial fluid and peritoneal effusion. Protein expression/identification was evaluated by 2-DE and MS analysis: six proteins showed differential expression in tumoral interstitial fluid and tumor tissue compared to normal interstitial fluid and healthy tissue: five were found to be downregulated and identified as galectin 3, glutathione S-transferase A-2, retinol binding protein 1, phosphatidylethanolamine-binding protein and annexin 5, while the calgranulin, was significantly upregulated in all pathological samples, including the ascitic fluid. Validation of S100A8 overexpression in carcinoma tissue was obtained by immunohistochemistry. To our knowledge, this is the first study to report an over-expression of calgranulin by 2-DE associated with MS/MS analysis on surgical biopsy. The reduced expression of galectin 3 and retinol binding protein 1 in cystic fluid and serum of patients with early stage disease is confirmed in this study. The results highlight alterations in proteins that control cell-cycle progression and apoptosis, as well as factors that modulate the activity of signal transduction pathways. Moreover, this study suggests that calgranulin expression may be used as a diagnostic and/or prognostic biomarker.

2009 - Identification of protein clusters predictive of response to chemotherapy in breast cancer patients [Articolo su rivista]
L., Cortesi; Barchetti, Andrea; DE MATTEIS, Elisabetta; Rossi, Elena; DELLA CASA, Lara; Marcheselli, Luigi; Tazzioli, Giovanni; M. G., Lazzaretti; G., Ficarra; Federico, Massimo; Iannone, Anna

An attempt for the identification of potential biomarkers predictive of response to chemotherapy (CHT) in breast cancer patients has been performed by the use of two-dimensional electrophoresis and mass spectrometry analysis. Since growth and progression of tumor cells depend also on stromal factors in the microenvironment, we choose to investigate the proteins secreted in Tumor Interstitial Fluid (TIF) and in Normal Interstitial Fluids (NIF). One-hundred and twenty-two proteins have been analyzed and a comparison was also made between the proteomic profile of responders versus nonresponders to CHT. At baseline, proteins isolated in TIF and NIF of all the 28 patients show significant differences in expression. Two clusters of proteins, differentially expressed in TIF with respect to NIF were found. Most significant is the decreased expression in TIF of CRYAB. In the protein metabolism group, also FIBB was found decreased. Some proteins involved in energy pathways were overexpressed (PGAM-1, ALDO A, PGK1, G3Pcn), while some other were down-regulated (CAH2, G3Pdx, PRDX6, TPIS). The same trend was observed for signal transduction proteins, with 14-3-3-Z overexpressed, and ANXA2 and PEBP 1 down-regulated. Moreover, an analysis has been conducted comparing protein expression in interstitial fluids of responders and nonresponders, irrespective of TIF or NIF source. This analysis lead us to identify two clusters of proteins with a modified expression, which might be predictive of response to CHT. In responders, an increase in expression of LDHA, G3Pdx, PGK1sx (energy pathways), VIME (cell growth and maintenance) and 14-3-3-Z (signal transduction), coupled with a decreased expression of TPIS, CAH 2, G3Psx, PGK 1dx (energy pathways), TBB5 (cell growth and maintenance), LDHB and FIBB (protein metabolism), was found. We observed that CHT modifies the expression of these cluster proteins since, after treatment, their expression in TIF of responder is generally decreased. Patients not responding to CHT show an unchanged expression pattern in TIF, with the exception of protein 14-3-3-Z, which is overexpressed, and a decreased expression in NIF of several cluster proteins. In conclusion, the identification of protein clusters associated with response to CHT might be important for predicting the efficacy of a specific antineoplastic drug and for the development of less empiric strategies in choosing the therapy to be prescribed to the single patient

2009 - Impairment of 8-iso-PGF(2ALPHA) isoprostane metabolism by dietary conjugated linoleic acid (CLA) [Articolo su rivista]
Iannone, Anna; A., Petroni; E., Murru; L., Cordeddu; G., Carta; M. P., Melis; Bergamini, Stefania; DELLA CASA, Lara; L., Cappiello; R., Carissimi; M., O'Shea; D., Bell; E., De Santis; S., Banni

8-iso-PGF(2alpha) isoprostane (IP) is one of the most-used markers of lipid peroxidation in experimental models and humans. After its formation, it is promptly metabolized to 2,3 dinor (DIN) in peroxisomes. Conjugated linoleic acid (CLA) is preferentially beta-oxidized in peroxisomes which may compete with IP, and thereby may affect its metabolism. In order to verify whether CLA is able to influence IP formation and/or metabolism and to explain the mechanism, we challenged rats supplemented with CLA or with triolein (as a control fatty acid), with a single dose of carbon tetrachloride (CCl(4)) or of bacterial lipopolysaccharide (LPS). The results showed that IP and its precursor arachidonic acid hydroperoxide, as well as malondialdehyde (MDA), increase significantly in the liver of rats challenged with CCl(4), irrespective of the diet, while in LPS-treated rats only nitrites in liver and isoprostane in plasma increase. On the other hand, the peroxisomal beta-oxidation products of IP, the DIN, is significantly lower in the CLA group with respect to control and triolein groups. To further investigate whether this is due to competition between CLA and IP at the cellular level, we incubated human fibroblasts from healthy subjects or patients with adrenoleukodystrophy (ALD), with CLA and/or commercially available IP. The rationale of this approach is based on the deficient peroxisomal beta-oxidation of fibroblasts from ALD patients, leading to a reduced formation of DIN. In both normal and ALD cells, the presence of CLA significantly inhibits the formation of DIN from IP. We may conclude that both in vitro and in vivo studies strongly suggest that CLA may impair IP catabolism in peroxisomes. Consequently an increase of IP, as a sole result of CLA intake, cannot be considered as a marker of lipid peroxidation.

2008 - Long-term n-acetylcysteine and l-arginine administration reduces endothelial activation and systolic blood pressure in hypertensive patients with type 2 diabetes [Articolo su rivista]
V., Martina; A., Masha; V. R., Gigliardi; L., Brocato; E., Manzato; A., Berchio; P., Massarenti; F., Settanni; DELLA CASA, Lara; Bergamini, Stefania; Iannone, Anna

OBJECTIVE: Reactive oxygen and nitric oxide (NO) have recently been considered to be involved in the cardiovascular complications of patients with type 2 diabetes, as NO is thought to lose its beneficial physiological effects in the presence of oxygen radicals. For this reason, we tested the effects of l-arginine (ARG) and N-acetylcysteine (NAC) administration in increasing NO bioavailability by reducing free radical formation. RESEARCH DESIGN AND METHODS: A double-blind study was performed on 24 male patients with type 2 diabetes and hypertension divided into two groups of 12 patients that randomly received either an oral supplementation of placebo or NAC + ARG for 6 months. RESULTS: The NAC + ARG treatment caused a reduction of both systolic (P < 0.05) and diastolic (P < 0.05) mean arterial blood pressure, total cholesterol (P < 0.01), LDL cholesterol (P < 0.005), oxidized LDL (P < 0.05), high-sensitive C-reactive protein (P < 0.05), intracellular adhesion molecule (P < 0.05), vascular cell adhesion molecule (P < 0.01), nitrotyrosine (P < 0.01), fibrinogen (P < 0.01), and plasminogen activator inhibitor-1 (P < 0.05), and an improvement of the intima-media thickness during endothelial postischemic vasodilation (P < 0.02). HDL cholesterol increased (P < 0.05). No changes in other parameters studied were observed. CONCLUSIONS: NAC + ARG administration seems to be a potential well-tolerated antiatherogenic therapy because it improves endothelial function in hypertensive patients with type 2 diabetes by improving NO bioavailability via reduction of oxidative stress and increase of NO production. Our study's results give prominence to its potential use in primary and secondary cardiovascular prevention in these patients.

2008 - Proteomic analysis of early urinary biomarkers of renal changes in type 2 diabetic patients [Articolo su rivista]
Bellei, Elisa; Rossi, Elena; Lucchi, L; Uggeri, S; Albertazzi, Alberto; Tomasi, Aldo; Iannone, Anna

Diabetic nephropathy (DN) is a complication associated with diabetes, leading to end-stage renal disease (ESRD). Despite significant progress in understanding DN, the cellular mechanisms leading to the renal damage are incompletely defined. In this study, with the aim to identify urine biomarkers for the early renal alterations in type 2 diabetes mellitus (T2D), we performed urinary proteomic analysis of 10 normoalbuminuric patients with T2D, 12 patients with type 2 DN (T2DN), and 12 healthy subjects. Proteins were separated by 2-DE and identified with ESI-Q-TOF MS/MS. Comparing the patients proteomic profiles with those of normal subjects, we identified 11 gradually differently changed proteins. The decreased proteins were the prostatic add phosphatase precursor, the ribonuclease and the kallikrem-3. Eight proteins were progressively increased in both patients groups: transthyretin precursor, Ig K chain C region, Ig K chain V-II region Cum, Ig K-chain V-III region SIE, carbonic anhydrase 1, plasma retinol-binding protein, β-2-microglobulin precursor, β-2-glycoprotein 1. The proteomic analysis allowed us to identify several increased urinary proteins, not only in T2DN but also in T2D patients in which the microalbuminuria was in the normal range. These patterns of urinary proteins might represent a potential tool for a better understanding of diabetic renal damage.

2007 - Metabolism of 8-iso-PGF2 alpha and conjugated linoleic acid (CLA) in vivo and in X-adrenoleukodystrophy fibroblast. [Abstract in Rivista]
Petroni, A; Iannone, Anna; Cordeddu, L; Murru, E; Carta, G; Melis, Mp; Bergamini, Stefania; Blasevich, M; Carissimi, R; Oshea, M; DE SANTIS, E; Banni, S.

not available

2007 - Metabolism of 8-iso-PGF2 alpha isoprostane is impaired by conjugated linoleic acid (CLA) [Abstract in Rivista]
Oshea, M; Iannone, Anna; Petroni, A; Murru, E; Cordeddu, L; Carta, G; Melis, Mp; Bergamini, Stefania; DELLA CASA, Lara; Blasevich, M; Carissimi, R; DE SANTIS, E; Banni, S.

not available

2007 - Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage [Articolo su rivista]
Giuliani, Daniela; C., Mioni; Bazzani, Carla; Zaffe, Davide; A. R., Botticelli; S., Capolongo; A., Sabba; M., Galantucci; Iannone, Anna; P., Grieco; E., Novellino; G., Colombo; Tomasi, Aldo; A., Catania; Guarini, Salvatore

Background and purpose: In circulatory shock, melanocortins have life-saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists. Experimental approach: Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. Key results: All shocked rats treated with saline died within 30-35 min. Treatment with NDP-alpha-MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg(-1) i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. Conclusions and Implications: Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock.

2006 - 2-D proteomic analysis of urine in diabetic patients with nephropathy [Abstract in Rivista]
Tomasi, Aldo; L., Lucchi; Uggeri, Simona; Bellei, Elisa; Rossi, Elena; Albertazzi, Alberto; Iannone, Anna

Mortality and morbidity associated with diabetes is diabetic nephropathy. We carried out a research project on the identification of specific proteomic profiles in patients with diabetic nephropathy. The rationale for this study is based on studies demonstrating that chronic inflammation and oxidative stress determine the complications observed in diabetic patients. Such a pathogenesis will determine post-translational protein modification, bringing to the identification of prognostic and predictive markers. Recent advances in proteomic profiling technologies have allowed preliminary profiling. Various specific proteomic profiles have been described for various pathologies, in particular tumors. Studies on proteomic profiling in diabetic nephropathy have not been performed yet. We analyzed easily available biological samples such as urine to evaluate the expression and function levels of urinary molecules that could become diagnostic, prognostic and predictive markers of disease.

2006 - Antioxidant effect of FeAOX-6 on free radical species produced during iron catalyzed breakdown of tert-butyl hydroperoxide [Articolo su rivista]
Rota, Cristina; Tomasi, Aldo; Palozza, P.; Manfredini, S.; Iannone, Anna

There is a body of evidences demonstrating, in biological systems, a cooperative interaction between tocopherols and carotenoids. FeAOX-6 is a novel antioxidant that combines the chroman head of alpha-tocopherol and a fragment of the isoprenyl chain of lycopene. We have tested its antioxidant effect on different radical species generated in a chemical system, where peroxyl, alkoxyl and methyl radicals are generated by the ferrous ion-mediated decomposition of tert-butyl hydroperoxide. We found that FeAOX-6 has the same effectiveness of alpha-tocopherol in quenching peroxyl radical with no contribution by lycopene. The antioxidant activity of FeAOX-6 on alkoxyl and methyl radicals is comparable to that of the equimolar mixture of the parent compounds. Lycopene is able to quench alkoxyl radical, while it has no effect on peroxyl radical, showing a different antioxidant activity compared to other carotenoids, such as beta-carotene and lutein.

2006 - Caratterizzazione di proteine nel tessuto e nel liquido interstiziale di neoplasie della mammella [Abstract in Atti di Convegno]
Barchetti, A; Cortesi, L; Rossi, E; Bellei, E; Tomasi, A; Iannone, A; Federico, M.

La classificazione dei tumori della mammella si basa attualmente sui parametri clinici e sulla morfologia cellulare; l’analisi immunoistochimica utilizza un numero ristretto di fattori prognostici e predittivi correlati a diverse funzioni cellulari come la proliferazione, l’angiogenesi, l’invasione e la metastatizzazione. Questi parametri si limitano a classificare i pazienti in sottogruppi con differente prognosi mentre l’uso di nuove tecnologie, quali lo studio delle proteine attraverso elettroforesi bidimensionale (2D), potrebbe portare ad un approccio terapeutico individualizzato e ad una ottimizzazione del trattamento. Lo scopo del nostro studio è quello di individuare nuovi markers nell’ambito dei tumori invasivi della mammella confrontando il profilo di espressione proteica di tessuto normale, patologico, di TIF (tumoral interstitial fluid) e di NIF (normal interstitial fluid). Nel microambiente tumorale si concentrano infatti un gran numero di molecole in parte secrete, in parte trasportate da vescicole di membrana o liberate in seguito a morte cellulare. Abbiamo ottenuto 8 biopsie mammarie da pazienti affette da carcinoma duttale infiltrante (G3) e carcinoma lobulare infiltrante (G2/3). L’indagine è stata condotta utilizzando circa 200 mg di tessuto mammario di carcinoma duttale e di tessuto adiacente sano. I frammenti di biopsia, sospesi in tampone PBS, sono stati incubati in CO2 5% a 37°C per un’ora e successivamente centrifugati 20 min. a 3000g. Da questa coltura è stato recuperato e analizzato il surnatante contenente le proteine rilasciate dal tessuto. Successivamente l’estrazione proteica è stata effettuata anche sul pellet di tessuto bioptico polverizzato in azoto e sonicato in tampone di lisi. Per ciascuna analisi 100ug di proteine sono state sottoposte ad elettroforesi 2D e visualizzate con metodica silver staining. Gli spots di interesse sono stati caratterizzati con la tecnica HPLC-ESI-MS/MS in seguito a purificazione e digestione con tripsina. L’analisi preliminare delle mappe bidimensionali attraverso il programma PDQuest (BIO-RAD) ha evidenziato spots proteici la cui intensità variava significativamente nei campioni tumorali. Da segnalare un’aumentata espressione dell’oncogene mitogeno DJ-1(PARK 7), coinvolto in pathways di trasduzione del segnale Ras dipendenti e delle proteine LYPA (Acyl-protein thioesterase) e LDHB (lactate dehydrogenase B) la cui overespressione è stata segnalata in linee cellulari di tumore mammario resistenti al trattamento anti-estrogenico. È stato inoltre riscontrato un aumentato livello di PSA1 (Prostatic Specific Antigen), una proteasi serinica associata in letteratura ad un basso indice di proliferazione e ad una prognosi favorevole. L’analisi e il confronto di un maggiore numero di casi permetterà di chiarire il significato e di ottenere informazioni specifiche sul pattern di espressione genica dei differenti stadi e istotipi del carcinoma mammario. Inoltre il confronto con pattern proteici sierici potrebbe essere di aiuto nell’identificazione di nuovi e predittivi marker ematici di neoplasia.

2006 - Comparative antioxidant activity of tocotrienols and the novel chromanyl-polyisoprenyl molecule FeAox-6 in isolated membranes and intact cells [Articolo su rivista]
Palozza, P; Verdecchia, S; Avanzi, L; Vertuani, S; Serini, S; Iannone, Anna; Manfredini, S.

Oxidative stress plays a pivotal role in the pathogenesis of several chronic diseases and antioxidants may represent potential tools for the prevention of these diseases. Here, we investigated the antioxidant efficiency of different tocotrienol isoforms (e-, delta-, gamma-tocotrienols), and that of FeAox-6, a novel synthetic compound which combines, by a stable covalent bond, the chroman head of vitamin E and a polyisoprenyl sequence of four conjugated double bonds into a single molecule. The antioxidant efficiency was evaluated as the ability of the compounds to inhibit lipid peroxidation, reactive oxygen species (ROS) production, heat shock protein (hsp) expression in rat liver microsomal membranes as well as in RAT-1 immortalized fibroblasts challenged with different free radical sources, including 2,2'-azobis(2-amidinopropane) (AAPH), tert-butyl hydroperoxide (tert-BOOH) and H2O2. Our results show that individual tocotrienols display different antioxidant potencies. Irrespective of the prooxidant used, the order of effectiveness was:delta-tocotrienol > gamma-tocotrienol = e-tocotrienol in both isolated membranes and intact cells. This is presumably due to the decreased methylation of delta-tocotrienol chromane ring, which allows the molecule to be more easily incorporated into cell membranes. Moreover, we found that FeAox-6 showed an antioxidant potency greater than that of delta-tocotrienol. Such an efficiency seems to depend on the concomitant presence of a chromane ring and a phytyl chain in the molecule, which because of four conjugated double bonds, may induce a greater mobility and a more uniform distribution within cell membrane. In view of these results, FeAox-6 represents a new potential preventive agent in chronic diseases in which oxidative stress plays a pathogenic role.

2006 - Detection of a stable nitroxide during chemical depolymerization of heparin [Articolo su rivista]
Rota, Cristina; L., Liverani; F., Spelta; G., Mascellani; Tomasi, Aldo; Iannone, Anna

The use of low-molecular-weight heparins (LMWHs) has become common, since compared to unfractionated heparin (UFH), they have a much longer plasma half-life and lower incidence of side effects. LMWHs are derived from the depolymerization of UFH, obtained either chemically, physically or enzymatically. We employed electron spin resonance (ESR) spectroscopy to study the depolymerization of UFH by copper in the presence of hydrogen peroxide. A stable nitroxide radical was detected. This could be generated by the hydroxyl radical attack either to the N-SO3- group or to free amino groups present in the UFH preparation.

2006 - Markers urinari di nefropatia diabetica [Poster]
Bellei, E; Rossi, E; Lucchi, L; Uggeri, S; Albertazzi, A; Tomasi, A; Iannone, A

Il diabete, come riportato ampiamente in letteratura, è una delle principali cause di insufficienza renale in stadio terminale (ESRD). Nonostante i progressi raggiunti nella comprensione della nefropatia diabetica, i meccanismi patogenetici che inducono il danno non sono ancora ben definiti. Alcuni studi hanno evidenziato come l’infiammazione cronica e i danni ossidativi ad essa correlati contribuiscano in modo determinante alle complicanze osservate in questi pazienti. Si può inoltre ipotizzare la presenza di modificazioni post-traduzionali di alcune proteine, che potrebbero essere impiegate come biomarkers prognostici e predittivi di malattia. Profili proteomici altamente specifici sono stati caratterizzati per varie patologie, soprattutto tumorali, ma indagini su profili proteomici nella nefropatia diabetica umana non sono tuttora presenti in letteratura. In questo studio abbiamo confrontato pattern proteici di soggetti sani con quelli di pazienti con nefropatia diabetica, cercando di individuare quadri proteici caratteristici dei diversi stadi di patogenesi, nonché modifiche post-traduzionali peculiari o mutazioni correlate alla patologia in esame. Partendo da liquidi biologici facilmente reperibili e maneggiabili come le urine, è possibile infatti valutare, mediante elettroforesi bidimensionale (2-DE), la funzionalità e i differenti livelli di espressione di molecole urinarie. Sono stati selezionati 20 pazienti con diabete di tipo II associato a nefropatia (valori di creatininemia > 1,5 mg/dl e presenza di microalbuminuria). Alle urine, immediatamente dopo la raccolta, è stato aggiunto un cocktail di inibitori delle proteasi. Una aliquota di ciascun campione è stata precipitata in acetone a -20°C, centrifugata e il pellet risospeso in un opportuno tampone. Dopo aver misurato la concentrazione proteica totale tramite un kit commerciale, 10 microgrammi di proteine sono stati utilizzati per l’analisi bidimensionale. I gel ottenuti sono stati colorati con metodica Silver stain massa-compatibile, gli spots di interesse trattati con tripsina e i peptidi estratti identificati tramite spettrometria ESI-LC- MS/MS. I risultati preliminari hanno evidenziato un aumento dei livelli delle seguenti proteine nelle urine dei pazienti diabetici nefropatici: albumina, transferrina e immunoglobuline G (IgG), che dalla letteratura risultano essere indici di danno glomerulare; alpha-1-microglobulina e alpha-1-antitripsina, indicatori di alterata funzionalità tubulare. Inoltre altre tre glicoproteine (zinc-alpha-2-glicoprotein, alpha-1-acid-glicoprotein e leucine-rich-alpha-2-glicoprotein), risultano quantitativamente aumentate. La presenza invece di altri due indicatori di danno tubulare, ovvero beta-2-microglobulina (b-2MG) e retinol binding protein (RBP), oltre alla proteina di membrana perlecan, è stata riscontrata solamente nelle urine dei pazienti e non in quelle dei soggetti di controllo. In conclusione, la 2-DE accoppiata alla spettrometria di massa potrebbe rappresentare un metodo rapido, sensibile e non-invasivo per identificare markers precoci di patologia renale.

2006 - Ricerca di biomarkers nel tumore ovarico mediante analisi proteomica [Abstract in Atti di Convegno]
Rossi, E; Cortesi, L; Barchetti, A; Bellei, E; Monari, E; Tomasi, A; Iannone, A; Abrate, M; Federico, M.

Il carcinoma ovarico rappresenta la quarta causa di morte nel sesso femminile in Europa e negli Stati Uniti. Nell’80 % dei casi infatti, la diagnosi avviene in uno stadio avanzato poiché la maggior parte delle neoplasie ovariche rimane clinicamente asintomatica negli stadi I e II. La sola terapia chirurgica assicura una sopravvivenza a cinque anni del 90 % delle pazienti diagnosticate allo stadio I, percentuale che si riduce al 35% negli stadi più avanzati. Il nostro obbiettivo è quello di utilizzare l’elettroforesi bidimensionale (2DE) e la spettrometria di massa per identificare e caratterizzare nuovi specifici biomarkers tumorali ovarici che permettano di effettuare una diagnosi precoce, di monitorare la progressione tumorale e di impostare una terapia mirata. Le indagini preliminari sono state effettuate su campioni bioptici di tessuto ovarico sano e tumorale, e su liquido proveniente da lavaggio peritoneale. Le proteine sono state estratte da circa 100 mg di tessuto congelato in azoto liquido, polverizzato in un mortaio e risospeso in tampone di lisi contenente urea, tiourea, CHAPS, Tris-HCl, ampholine pH 3-10 e inibitori delle proteasi. Dopo incubazione a temperatura ambiente per un’ora, i campioni sono stati sonicati e centrifugati. Il pellet, ottenuto dopo precipitazione in acetone freddo, è stato risospeso in un buffer di reidratazione (urea, tiourea, CHAPS, DTT e ampholine) e il contenuto proteico totale misurato con il metodo Bradford. L’isoelettrofocalizzazione (IEF) è stata effettuata caricando 80 g di proteine totali su IPG strips a range di pH 3-10 non lineare, fino al raggiungimento di 70000 Vh finali. Le proteine focalizzate sono state ridotte con DTT e alchilate con Iodoacetamide in tampone di equilibrazione (Urea, Tris-HCl, Glicerolo, SDS, Blu di Bromofenolo) prima della separazione elettroforetica su gels di poliacrilamide (SDS–PAGE), a concentrazione omogenea 10% e a gradiente 8-16%. I gel sono stati infine colorati con metodica Silver-staining massa-compatibile. L’analisi con il programma PDQuest ha evidenziato la presenza di numerosi spot proteici differenzialmente espressi nei campioni tumorali rispetto ai controlli sani; la spettrometria di massa ESI-Q-TOF-MS/MS porterà all’identificazione delle proteine di interesse. Per quanto riguarda il liquido derivato da lavaggio peritoneale, prima dell’analisi 2DE le aliquote prelevate sono state concentrate con filtri Amicon aventi cut-off di PM 5 KDa. Parallelamente alla 2DE, sui campioni tissutali è stata effettuata anche l’analisi con tecnologia SELDI-TOF (Surface Enhanced Laser Desorption Ionisation-Time of Flight). I risultati ottenuti hanno evidenziato variazioni significative dell’espressione di numerose proteine, soprattutto a basso peso molecolare (<20 KDa). Lo scopo dello studio ha la finalità di giungere all’identificazione di biomarcatori specifici del tumore ovarico che possono essere evidenziati anche nel liquido peritoneale ed eventualmente nel siero, fungendo da fattori prognostici della malattia ed anche predittivi di risposta al trattamento con derivati del platino.

2006 - Ricerca di markers diagnostici, prognostici e predittivi in carcinomi mammari attraverso analisi proteomica [Abstract in Atti di Convegno]
Pecorari, L; Silvestri, C; Candini, O; Rossi, E; Bellei, E; Bussolari, R; Iannone, A; Federico, M; Cortesi, L; Calabretta, B.

Le basi molecolari dei tumori sporadici della mammella sono solo in parte conosciute: un comune meccanismo molecolare, che è stato riportato in circa il 75% dei casi, è l’attivazione della proteina oncogenica ad attività Serin/Treonin chinasica AKT. Una delle principali funzioni di pAKT (la cui forma attiva è fosforilata) è quella di promuovere la sopravvivenza cellulare mediata da fattori di crescita bloccando i meccanismi intrinseci dell’apoptosi. Recentemente numerosi studi si sono concentrati sull’approfondimento del ruolo dell’oncoproteina AKT nel carcinoma mammario; la conclusione comune a questi studi è che si possano ottenere benefici clinici rilevanti dall’appropriata combinazione terapeutica tra chemioterapici convenzionali e inibitori specifici di pAKT. Sarebbe inoltre di notevole importanza chiarire in che modo la valutazione dell’attività di pAKT possa essere interpretata ai fini diagnostici, prognostici e predittivi in pazienti con carcinoma mammario. Questo progetto di ricerca si propone di approfondire questi aspetti utilizzando un approccio basato su tecniche di analisi proteomica. Su diversi tipi di linee continue di carcinoma mammario con diverse caratteristiche e diverso grado di aggressività (ErbB2+ / ErbB2- / BRCA1 mutato / BRCA1 w.t.) sono stati eseguiti e sono tuttora in corso, esperimenti d’inibizione/deplezione della proteina pAkt. In particolare dalla linea SKBr3 è stato possibile ottenere estratti proteici contenenti una discreta quota di pAKT; inoltre esperimenti di immunoprecipitazione selettiva hanno consentito di isolare gli interattomi di pAKT e di AKT non fosforilata. Questi immunoprecipitati sono poi stati sottoposti ad elettroforesi bidimensionale e gli spot indicativi di proteine varianti (associate quindi ad una forma rispetto all’altra) analizzati tramite spettrometria di massa. Attualmente una sola nuova potenziale proteina cliente, Elongation Factor 1 alfa (EF1a), è stata identificata ed è in via di caratterizzazione. Per approfondire il ruolo di questa proteina (nota per essere iperespressa in diversi tipi di carcinoma tra i quali l’ovarico, il mammario e il prostatico) sono stati condotti studi di “RNAinterference” che hanno permesso di asserire che in cellule di carcinoma mammario la riduzione dell’espressione di EF1a (livelli diminuiti del 60%) porta ad una riduzione del 30% della clonogenicità. Sembra inoltre che diminuiti livelli d’espressione di EF1 associati a trattamenti a base di 17AAG (inibitore selettivo dell’attività di HSP90 che causa forti riduzioni ai livelli di pAKT) abbiano un effetto sinergico nella riduzione della proliferazione cellulare. Parallelamente a questi studi, stiamo programmando un’analisi globale di tutte le proteine che manifestano una espressione differenziale dopo l’inibizione di pAKT (globale, perché non si riferisce alle sole proteine dell’interattoma, ma all’estratto totale). Questo verrà realizzato tramite confronto diretto delle mappe proteiche ottenute dopo elettroforesi bidimensionale degli estratti totali (trattamento inibitorio specifico vs. controllo) individuando e identificando gli “spots” che rappresentano proteine la cui espressione varia a seguito dell’inibizione dell’attività chinasica di pAKT. La scoperta di nuove proteine substrato della attività chinasica di pAKT e l’approfondimento dei meccanismi biologici e molecolari da essa regolati, rappresentano un importante punto di partenza per la progettazione di strategie terapeutiche ottimali e per la validazione di nuovi markers diagnostici, prognostici o predittivi in situazioni di carcinoma mammario.

2006 - Role of oxidative stress in progressive kidney failure [Abstract in Rivista]
Tomasi, Aldo; S., Uggeri; Bergamini, Stefania; DELLA CASA, Lara; Albertazzi, Alberto; L., Lucchi; Iannone, Anna

not available

2006 - α-Tocopherol amplifies benzoyl peroxide free radical decomposition in a chemical system [Articolo su rivista]
Rota, Cristina; Tomasi, Aldo; Iannone, Anna

Benzoyl peroxide is commonly used in the treatment of acne, even though some adverse effects have been reported, probably mediated by the formation of peroxide-derived free radicals and the depletion of antioxidants. In the present work we have studied, in a chemical system, the effect of a-tocopherol on benzoyl peroxide radical decomposition to analyse the presence of an interaction between these two compounds, leading to an enhanced peroxide-cytotoxicity, as we have previously reported. Under our experimental conditions alpha-tocopherol strongly amplified the peroxide free radical decomposition occurring either in the presence or in the absence of UV irradiation, and lead to the formation of an unknown radical species in addition to benzoyloxy, phenyl and tocopheroxyl free radicals. The results of this study show that the enhancement of benzoyl peroxide toxicity in cells exposed simultaneously to this peroxide and alpha-tocopherol, is likely due to the generation of the detected radical species.

2005 - Activation of an efferent cholinergic pathway produces strong protection against myocardial ischemia/reperfusion injury in rats [Articolo su rivista]
Mioni, C.; Bazzani, Carla; Giuliani, Daniela; Altavilla, D.; Leone, S.; Ferrari, Anna; Minutoli, L.; Bitto, A.; Marini, H.; Zaffe, Davide; Botticelli, Ar; Iannone, Anna; Tomasi, Aldo; Bigiani, Albertino; Bertolini, Alfio; Squadrito, F.; Guarini, Salvatore

Objective: A vagus nerve-mediated, brain cholinergic protective mechanism activated by melanocortin peptides is operative in conditions of circulatory shock; moreover, there is anatomical evidence of dual vagal-cardiac efferent pathways in rats, which could play different roles in controlling heart function. Therefore, we investigated the role and functional mechanism of such vagal efferent pathway(s) in an experimental model of ischemic heart disease. Design: Randomized experimental study. Setting: Research laboratory. Subjects: Adult Wistar rats of either sex. Interventions: After bilateral cervical vagotomy (with or without pretreatment with atropine), efferent vagal fibers were electrically stimulated in rats subjected to coronary artery occlusion (5 mins) followed by reperfusion (5 mins). Other rats (intact, vagotomized, or pretreated with atropine) were treated with nanomolar doses of melanocortin peptides. Measurements and Main Results: Electrical stimulation of efferent vagal fibers (5 V, 2 msecs,1-9 Hz, for the whole period of ischemia/reperfusion) strongly reduced the high incidence of severe arrhythmias and lethality, reduced the increase in free radical blood levels and left-ventricle histologic alterations, and augmented the extracellular signal-regulated kinase activation. Treatment with the melanocortin peptides adrenocorticotropin and gamma(2)-melanocyte-stimulating hormone (162 nmol/kg intravenously or 16.2 nmol/kg intracerebroventricularly, during coronary occlusion) produced the same protective effects of electrical stimulation and with the same muscarinic acetylcholine receptor-dependent mechanism, seemingly through brain activation (mediated by melanocortin MC3 receptors, as previously described) of such efferent vagal pathway. Conclusions: The present results give evidence for the identification of a protective, melanocortin-activated, efferent vagal cholinergic pathway, operative in conditions of myocardial ischemia/reperfusion. These data suggest that melanocortins and pertinent compounds able to activate such a pathway could provide the potential for development of a new class of drugs for a novel approach to management of ischemic heart disease.

2005 - Changes in conjugated linoleic acid and palmitoleic acid are correlated to retinol levels in chronic renal failure in both hemodialysis and conservative treatment patients [Articolo su rivista]
L., Lucchi; S., Banni; Iannone, Anna; Mp, Melis; G., Carta; E., Murru; L., Cordeddu; L., Stipo; S., Uggeri; V., Gatti; V., Malaguti; Albertazzi, Alberto

An increase in conjugated linoleic acid (CLA), a natural fatty acid present in our diet, which possesses anticarcinogenic and antiatherogenic activities in experimental models, has been found in both the plasma and adipose tissue of end-stage chronic renal failure (ESCRF) patients. Increased levels of retinol have also been found in those patients, due to a reduced excretion of the retinol-binding protein. Since retinol is known to influence lipid metabolism, we evaluated whether changes in retinol, CLA, and other fatty acids are correlated in the plasma of CRF patients. We measured CLA, retinol, and unsaturated fatty acids in the plasma of the following groups: (A) 35 ESCRF patients; (B) 20 hemodialysis (HD) patients; (C) 20 healthy controls. Subjects with total cholesterol and/or triglycerides higher than 250 mg/dL were excluded. We found a significant increase in CLA, retinol, palmitoleic (16:1), and oleic (18:1) acids in ESCRF patients. In HD patients we found a similar pattern, however, CLA increase was not significant. No changes were observed in the other fatty acids measured. In the groups of ESCRF and HD patients, a positive correlation between the levels of plasma retinol and CLA, and between retinol and 16:1 was found. These correlations were not detected in controls. The abnormal levels of plasma retinol in CRF patients might partly explain the changes in CLA and 16:1. The influence of retinol levels on these fatty acids might be due to an induction of delta 9 desaturase. In fact, 16:1 is known to be produced, partly, by delta 9 desaturation of palmitic acid. Moreover, the formation of CLA from delta 9 desaturation of vaccenic acid-a trans-monounsaturated fatty acid present in our diet-has recently been demonstrated in humans. Nevertheless, our data do not represent direct evidence supporting an increased delta 9 desaturase activity in CRF patients. Another possible explanation might be a variation in the exogenous intake.

2005 - Comparison between hydroperoxides and malondialdehyde as markers of acute oxidative injury during hemodialysis [Articolo su rivista]
L., Lucchi; Iannone, Anna; Bergamini, Stefania; L., Stipo; S., Perrone; S., Uggeri; V., Gatti; F., Ferrari; Tomasi, Aldo; Albertazzi, Alberto

An increased free-radical production has been documented during hemodialysis (HD) particularly when bio-incompatible membranes are utilized. These highly reactive free radicals can cause damage through several pathways, one of the best known being lipid peroxidation. Malondialdehyde (MDA) is a product of lipid peroxidation, which can partly be removed by HD due to its low molecular weight and water solubility. Hydroperoxides are predominantly found in lipid substances, and therefore their removal by HD could be difficult. We evaluated the behavior of these two by-products of lipid peroxidation during HD, comparing their behavior in three different membranes, in order to study their reliability as markers of acute oxidative injury. Fifteen stable HD patients were dialyzed with each of the following membranes: cuprophan, polyamide, and polysulfone, three sessions for every membrane. MDA and hydroperoxides were measured pre-HD and then both from the arterial and venous line at 8, 15, 30, and 240 min. During HD with cuprophan membrane MDA decreased significantly in the venous line compared with the arterial line at 8, 15, and 30 min (P < 0.05). At the end of HD, MDA was significantly reduced compared with MDA pre-HD (P < 0.05). Plasma hydroperoxides increased significantly in the venous line compared with the arterial line at 8, 15, 30, and 240 min (P < 0.05). At the end of HD, hydroperoxides had increased significantly as compared with pre-HD (P < 0.05). When the polyamide and polysulfone membranes were used, the behavior of MDA was similar to that found with cuprophan. Hydroperoxides were unchanged during HD using both membranes. MDA is not a reliable marker of acute oxidative injury during HD as it is removed during HD. Hydroperoxide measurement is a better marker of acute oxidative injury during HD.

2005 - Effetto della N-acetilcisteina sulla progressione dell'insufficienza renale cronica nel ratto [Poster]
Bellei, E; Bergamini, S; Ligabue, G; Ballestri, M; Albertazzi, A; Tomasi, A; Iannone, A.

L’insufficienza renale cronica porta nel tempo ad un progressivo deterioramento della funzionalità renale, sino ad arrivare a quella che viene definita come “end-stage renal disease” (ESRD). I meccanismi coinvolti in questo processo multifattoriale sono mediati dall’azione di diverse sostanze, fra cui: angiotensina II, TGF, endotelina, monossido d’azoto (NO), radicali liberi dell’ossigeno e citochine. Si è voluto indagare l’effetto di una dieta arricchita con una sostanza antiossidante e anti-infiammatoria, la NAC, sulla progressione dell’insufficienza renale cronica indotta in ratti mediante asportazione chirurgica dei 5/6 del parenchima renale.

2005 - Erythrocyte susceptibility to oxidative stress in chronic renal failure patients under different substitutive treatments [Articolo su rivista]
Lucchi, L; Bergamini, Stefania; Iannone, Anna; Perrone, S; Stipo, L; Olmeda, F; Caruso, F; Tomasi, Aldo; Albertazzi, Alberto

An increased oxidative stress is now considered one of the major risk factors in chronic renal failure (CRF) patients that may be exacerbated by dialysis. It has been postulated that this increased oxidative stress might cause an augmented red blood cell (RBC) membrane lipid peroxidation with the consequent alteration in membrane deformability. The aim of this study was to evaluate RBC susceptibility to an in vitro induced oxidative stress and RBC antioxidant potential in different groups of CRF patients undergoing different substitutive treatment modalities. Fifteen end-stage CRF patients were evaluated in conservative treatment, 23 hemodialysis (HD) patients, 15 continuous ambulatory peritoneal dialysis (CAPD) patients, 15 kidney transplanted patients, and 16 controls. Their RBCs were incubated with the oxidative stress-inducing agent tert-butylhydroperoxide both in the presence and in the absence of the catalase inhibitor sodium azide, and the level of malondialdehyde (MDA) (a product of lipid peroxidation), was measured at 0, 5, 10, 15, and 30 min of incubation. In addition, the RBC content of reduced glutathione (GSH) was measured by HPLC. As opposed to the controls, RBCs from end-stage CRF patients exhibited an increased sensitivity to oxidative stress induced in vitro, both in the absence and presence of a catalase inhibitor, as demonstrated by a significantly higher level of MDA production at all the incubation times (P < 0.05). Different substitutive treatments had different impacts on this phenomenon; CAPD and kidney transplantation were able to normalize this alteration while HD was not. GSH appeared to be related to the increase in RBC susceptibility to oxidative stress; its content being significantly elevated in end-stage CRF and HD patients as compared with CAPD and transplanted patients and controls (P < 0.05). No significant changes were observed in the RBC glutathione content during the HD session. The increase of GSH in RBCs of end-stage CRF and HD patients seems to indicate the existence of an adaptive mechanism under increased oxidative stress occurring in vivo. Unlike HD, the beneficial effect of CAPD on the anemia of dialysis patients might partly be due to a condition of lower oxidative stress that might in addition counterbalance the cardiovascular negative effects of dislipidemia of CAPD patients.

2005 - Free radical generation during chemical depolymerization of heparin [Articolo su rivista]
Rota, Cristina; L., Liverani; F., Spelta; G., Mascellani; Tomasi, Aldo; Iannone, Anna; E., Vismara

Low-molecular weight heparins (LMWHs), as compared with unfractionated heparin (UFH), present superior bioavailability, much longer plasma half-life, and lower incidence of side effects. For these reasons, over the past two decades LMWHs have become the drugs of choice for the treatment of deep venous thrombosis, pulmonary embolism, arterial thrombosis, and unstable angina. Furthermore, their use in acute ischemic stroke is currently under study. LMWHs are obtained by UFH depolymerization, which can be performed using various methods, including nitrous acid depolymerization, cleavage by beta-elimination of benzyl ester, enzymatic depolymerization, and peroxyl radical-dependent depolymerization. This article addresses the chemical depolymerization, obtained by free radical attack (mainly hydroxyl radical), of heparin. The electron spin resonance (ESR) spectroscopy, coupled to the spin trapping technique, was employed to study this reaction. Free radical-mediated heparin depolymerization was performed under different chemical conditions. The final products of the reactions were purified and classified on the basis of their molecular weight and other characteristics. The level of heparin fragmentation was different depending on the type of depolymerization reaction used. Moreover, the level of reproducibility and the resulting radical species were different for every type of reaction performed.

2005 - No evidences of oxidative injury associated with slow intravenous administration of ferric gluconate during haemodialysis [Relazione in Atti di Convegno]
Bergamini, Stefania; DELLA CASA, Lara; Iannone, Anna; Albertazzi, Alberto

No evidences of oxidative injury associated with slow intravenous administration of ferric gluconate during haemodialysis

2005 - Oxidative stress and experimental shock [Abstract in Rivista]
Tomasi, Aldo; Bergamini, Stefania; Bellei, Elisa; Bazzani, Carla; Bertolini, Alfio; Guarini, Salvatore; Iannone, Anna

In this introductory presentation, some of the recent results of our and other groups on the oxidative stress theory in the development of experimental shock, will be presented and discussed.

2005 - Oxidative stress in renal patients [Abstract in Rivista]
Iannone, Anna; Bergamini, Stefania; Bellei, Elisa; Rota, Cristina; DELLA CASA, Lara; Tomasi, Aldo

non disponibile

2004 - A comparison between the antioxidant and peroxynitrite-scavenging functions of the vitamin E metabolites alpha- and gamma-carboxyethyl-6-hydroxychromans [Articolo su rivista]
F., Galli; M., Piroddi; Iannone, Anna; S., Pagliarani; Tomasi, Aldo; A., Floridi

Carboxyethyl-6-hydroxychromans (CEHC) are vitamin E metabolites with proposed in vitro antioxidant function. In this study we compared the antioxidant potency of the two main CEHC metabolites found in biological fluids (i.e., alpha-CEHC and gamma-CEHC) using two different experimental models of lipid oxidation: 1) plasma diluted 1/50 vol/vol in phosphate buffered saline (PBS) exposed to 50 muM Cu2+ ions, and 2) LDL (100 mug of proteins) exposed to different pro-oxidants as 2.5 muM Cu2+, 1 mM of the water soluble peroxyl radical generator 2,2'-Azobis(2-amidinopropane) hydrochloride (AAPH) and human macrophages (4 x 10(5) cells). Moreover, the two CEHC homologues were assessed for the inhibitory effect on the peroxynitrite (ONOO-)-induced nitration of tyrosine (Tyr). The results showed that in the concentration range 0.015-5 muM the CEHC metabolites and the hydrosoluble analogue Trolox exert similar concentration-dependent inhibition of the Cu2+-induced lipid oxidation of plasma. After in vitro exposure to tert-butyl hydroperoxide/Fe2+, CEHC formed chromanoxyl radicals with electron spin resonance spectra matching exactly those of their parent tocopherols. The LDL oxidation induced by AAPH or Cu2+ was significantly and similarly inhibited by 1 muM of both the CEHC homologues and Trolox. gamma-CEHC showed a slight but significantly higher inhibition of the macrophage-induced low-density lipoprotein (LDL) oxidation than alpha-CEHC. Both the CEHC homologues inhibit Tyr nitration induced by ONOO-. However, gamma-CEHC produced a slightly greater inhibitory effect than alpha-CEHC through the formation of the nitrated congener 5-nitro-gamma-CEHC. In all the systems under investigation, low nanomolar concentrations of CEHC (i.e., the concentration range in the blood of subjects with normal dietary intake of vitamin E) produced feeble antioxidant effects. In conclusion, gamma-CEHC and alpha-CEHC show similar concentration-dependent inhibition of plasma and LDL lipid oxidation. gamma-CEHC has a fairly higher potency than alpha-CEHC as ONOO- scavenger through the formation of 5-nitro-gamma-CEHC. CEHC metabolites show the same in vitro antioxidant chemistry of their parent tocopherols, but the characteristic hydrophilicity of these metabolites could result in different biopotency and roles. Further studies are needed to clarify whether CEHC could contribute to the antioxidant network in biological fluids and tissues.

2004 - Antiossidanti come modulatori del metabolismo degli acidi grassi [Abstract in Atti di Convegno]
Banni, S; Melis M., P; Carta, G; Cordeddu, L; Murru, E; Bellei, E; Iannone, A

Antiossidanti come modulatori del metabolismo degli acidi grassi

2004 - Chronic inflammation in end stage renal disease: markers of oxidative stress and redox modification [Abstract in Atti di Convegno]
Iannone, A; Bergamini, S; Bellei, E; Rota, C; Tomasi, A

Markers of oxidative stress and redox modification in patients with end stage renal disease and chronic inflammation

2004 - Effect of alpha-tocopherol and N-acetylcysteine on benzoyl peroxide toxicity in human keratinocytes [Articolo su rivista]
Bellei, Elisa; Rota, Cristina; Bergamini, Stefania; P., Manfredini; Albertazzi, Alberto; Tomasi, Aldo; Iannone, Anna

Benzoyl peroxide is a free-radical generating compound widely used in the polymer industry and also in pharmaceuticals as antimicrobial agent to treat acne. However, benzoyl peroxide causes irritation and contact dermatitis in about 1% of patients. Concern over the use of this compound is motivated by the demonstration that it can also act as skin tumor promoter in mice. In addition, benzoyl peroxide induces DNA strand breaks in many cells, including keratinocytes. Benzoyl peroxide toxicity is presumably mediated by the formation of reactive free radicals and by the consumption of intracellular antioxidants. In this work we investigated the effect of both the lipophilic antioxidant et-tocopherol and the hydrophilic thiol donor N-acetylcysteine (NAC) in human keratinocyte line HaCaT exposed to benzoyl peroxide. A protective effect against benzoyl peroxide cytotoxicity was achieved when cells were grown on a alpha-tocopherol layer. On the contrary, the addition of alpha-tocopherol dissolved in ethanol had a pro-oxidant effect, leading to an enhancement of benzoyl peroxide toxicity. Cytotoxicity was also reduced adding NAC to the culture medium; the presence of both NAC and alpha-tocopherol exerts a synergistic cytoprotection.

2004 - Effetti della N-acetilcisteina in condizioni di stress ossidativo [Abstract in Atti di Convegno]
Iannone, A; Bellei, E; Bergamini, S; Rota, C; Tomasi, A; Albertazzi, A.

Effetti della N-acetilcisteina in condizioni di stress ossidativo

2004 - Erratum: Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors (European Journal of Pharmacology (2003) 477 (227-234)) [Articolo su rivista]
Mioni, C.; Giuliani, D.; Cainazzo, M. M.; Leone, S.; Iannone, A.; Bazzani, C.; Grieco, P.; Novellino, E.; Tomasi, A.; Bertolini, A.; Guarini, S.

2004 - Markers of inflammation in end stage renal disease patients. A role for antioxidants [Abstract in Atti di Convegno]
Iannone, A; Bellei, E; Bergamini, S; Rota, C; Tomasi, A

Markers of inflammation in end stage renal disease patients. A role for antioxidants

2004 - Markers of oxidative stress and antioxidant status in haemodialysis patients [Relazione in Atti di Convegno]
Iannone, A; Bergamini, S; Bellei, E; Rota, C; Lucchi, L; Albertazzi, A; Tomasi, A

Study of oxidative stress markers and antioxidant status in haemodialysis patients

2004 - Redox regulation and antioxidant in shock [Abstract in Atti di Convegno]
Iannone, A; Bellei, E; Bergamini, S; Rota, C; Tomasi, A

Redox regulation and antioxidant in shock

2004 - Relationship of asymmetric dimethylarginine to haemodialysis hypotension [Articolo su rivista]
Bergamini, Stefania; L., Vandelli; Bellei, Elisa; Rota, Cristina; P., Manfredini; Tomasi, Aldo; Albertazzi, Alberto; Iannone, Anna

Hypotension is one of the major complications in patients undergoing haemodialysis (HD), that is well evident in patients defined as hypotension-prone. The mechanisms underlying the hypotensive episodes are not known. We carried out a clinical study on hypotension-prone HD patients to test the existence of a dysregulation in the nitric oxide (NO) generating pathway. Since asymmetric dimethylarginine (ADMA) is an endogenous compound which regulates NO synthesis, we measured its variation in plasma of stable-HD and hypotension-prone patients before, during, and at the end of HD. Before HD, the hypotension-prone patients have higher ADMA levels than stable-HD patients. The HD procedure significantly removes ADMA from plasma of stable-HD patients, while in the hypotension-prone ADMA levels are unchanged at the end of the HD. Moreover, in the hypotension-prone patients, during the hypotensive episode, a dramatic drop of ADMA levels is observed, followed by a rapid increase at the end of the HD. The symmetric dimethylarginine (SDMA), which has no effect on NO synthesis, is also high in plasma of both groups of HD patients compared to normal subjects, and in both groups its levels at the end of HD are significantly reduced. The hypotension-prone patients have basal TNF-alpha levels lower than the stable-HD groups, that significantly increase during the hypotensive episode. On the basis of these findings, we suggest that the hypotensive syndrome could be related to a dysregulation between ADMA metabolism and clearance due both to cytokines release and to an extremely fast ADMA clearance during HD, leading to an increase in NO blood levels.

2004 - Role of nitric oxide generating pathway in haemodialysis hypotension [Poster]
Bergamini, S; Rota, C; Bellei, E; Albertazzi, A; Vandelli, L; Tomasi, A; Iannone, A

Role of nitric oxide in haemodialysis hypotension

2003 - Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors [Articolo su rivista]
Mioni, Chiara; Giuliani, Daniela; Mm, Cainazzo; Leone, Sheila; Iannone, Anna; Bazzani, Carla; P., Grieco; E., Novellino; Tomasi, Aldo; Bertolini, Alfio; Guarini, Salvatore

In rats subjected to myocardial ischemia/reperfusion, melanocortin peptides, including gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), are able to exert a protective effect by stimulating brain melanocortin MC3 receptors. A non-melanocortin receptor belonging to a group of receptors for Phe-Met-Arg-Phe-NH2 (FMRFamide)-like peptides may be involved in some of the cardiovascular effects of the gamma-MSHs. FMRFamide-like peptides and gamma(1)-/gamma(2)-MSH share, among other things, the C-terminal Arg-Phe sequence, which seems to be essential for cardiovascular effects in normal animals. So we aimed to further investigate which receptor and which structure are involved in the protective effects of melanocortins in anesthetized rats subjected to myocardial ischemia by ligature of the left anterior descending coronary artery (5 min), followed by reperfusion. In saline-treated rats, reperfusion induced, within a few seconds, a high incidence of ventricular tachycardia and ventricular fibrillation, and a high percentage of death within the 5 min of observation period. Reperfusion was associated with a massive increase in free radical blood levels and with an abrupt and marked fall in systemic arterial pressure. The i.v. treatment (162 nmol/kg) during the ischemic period with the adrenocorticotropin fragment 1-24 [ACTH-(1-24): the reference protective melanocortin which binds all melanocortin receptors], as well as with both the melanocortin MC3 receptor agonists gamma(2)-MSH and [D-Trp(8)]gamma(2)-MSH, reduced the incidence of ventricular tachycardia, ventricular fibrillation and death, the increase in free radical blood levels and the fall in arterial pressure. On the contrary, gamma(2)-MSH-(6-12) (a fragment unable to bind melanocortin receptors) was ineffective. Such protective effect was prevented by the melanocortin MC3/MC4 receptor antagonist SHU 9119. In normal (i.e., not subjected to myocardial ischemia/reperfusion) rats, the same i.v. dose (162 nmol/kg) of gamma(2)-MSH, [D-Trp(8)]gamma(2)-MSH and gamma(2)-MSH-(6-12) provoked a prompt and transient increase in arterial pressure; on the other hand, ACTH-(I -24), which lacks the C-terminal Arg-Phe sequence, decreased arterial pressure, but only at higher doses. Heart rate of normal rats was not affected by any of the assayed peptides. The present data confirm and extend our previous findings that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors. Moreover, they further support the notion that, in normal rats, cardiovascular effects of gamma-MSHs are mediated by receptors for FMRFamide-like peptides, for whose activation, but not for that of melanocortin MC3 receptors, the C-terminal Arg-Phe structure being relevant.

2003 - Measuring oxidative stress and redox state in patients: meaning and perspectives [Poster]
Tomasi, A; Bergamini, S; Bellei, E; Rota, C; Iannone, A.

Measuring oxidative stress and redox state in patients with ESRD.

2003 - Nitric oxide and hypotension in haemodialysis patients. [Abstract in Rivista]
Iannone, Anna; Bellei, Elisa; Bergamini, Stefania; Rota, C.; Vandelli, L.; Albertazzi, Alberto; Tomasi, Aldo


2003 - Plasma antioxidants modifications during haemodialysis [Poster]
Bellei, E; Manfredini, P; Lucchi, L; Bergamini, S; Rota, C; Tomasi, A; Vandelli, L; Albertazzi, A; Banni, S; Iannone, A.

The high incidence of atherosclerosis (stroke and ischemic heart disease) in patients with end-stage renal failure (ESRF), associated with clinical and/or laboratory signs of systemic inflammation, has been considered to be in part due to the long-term haemodialysis (HD). The contact with the artificial surface of the extra-corporeal circuit may promote free radicals formation, which is responsible for the increased lipid peroxidation observed in these patients. It has been also demonstrated that they have a decreased intracellular antioxidant capability. To test the presence of oxidative stress and evaluate also the antioxidant status during the dialytic session, we performed a clinical study in a group of functionally anephric patients on maintenance HD treatment with three weekly HD sessions, using a low-flux cellulose diacetate membrane and a freshly prepared bicarbonate buffer.

2003 - Redox regulation, cytokines, and nitric oxide in inflammation [Capitolo/Saggio]
Tomasi, Aldo; Bergamini, Stefania; Rota, C.; Iannone, Anna

non disponibile

2003 - “Role of NO in hypotension prone dialytic patients” [Abstract in Rivista]
Iannone, Anna; Bellei, Elisa; Bergamini, Stefania; Rota, Cristina; Vandelli, Lorenza; Albertazzi, Alberto; Tomasi, Aldo

not available

2002 - Antioxidants modulates benzoyl-peroxide toxicity in human keratinocytes [Abstract in Rivista]
Manfredini, P; Bellei, E; Bergamini, S; Rota, C; Iannone, A; Tomasi, A; Albertazzi, A.

Benzoyl peroxide (BP) is a free-radical generating compound, widely used in the polymer industry and also in pharmaceuticals as antimicrobial agent to treat acne. However, BP causes irritation and contact dermatitis in about 1% of patients. Concern over the use of this compound is motivated by the demonstration that it can also act as skin tumor promoter in mice. In addition, BP induces DNA strand breaks in many cells, included keratinocytes. In vitro, the tossicity associated with BP is presumably mediated by formation of benzoyloxyl radical, responsable for membrane damage. In this work we investigated the effect of both the lipophilic antioxidant a-tocopherol and the thiol donor N-acetylcysteine (NAC) in human keratinocyte line HaCaT exposed to BP.

2002 - N-Acetylcysteine negatively modulates nitric oxide production in endotoxin-treated rats through inhibition of NF-ĸB activation [Articolo su rivista]
C., Rota; Bergamini, Stefania; F., Daneri; Tomasi, Aldo; F., Virgili; Iannone, Anna

N-Acetylcysteine (NAC) has a wide spectrum of biological activities, either related to the ability to increase intracellular thiols or directly acting as an antioxidant. We used an in vivo animal model to study NAC modulation of nitric oxide (NO) production in response to lipopolysaccharide treatment. A comparison was made between NAC and the N-[3-(aminomethyl)benzyl] acetamidine (1400W), an inhibitor of the inducible NO synthase (iNOS). Both inhibit NO production, although NAC lacks any effect if given when iNOS is already induced; this indicates that the decrease of NO generation is not due to an effect on iNOS activity. We found that the DNA binding activity of nuclear transcription factor-kappaB in peripheral blood cells was inhibited by NAC given before lipopolysaccharide, whereas tumor necrosis factor-a secretion was not affected.

Manfredini, P; Bellei, Elisa; Bergamini, Stefania; Rota, Cristina; Tomasi, Aldo; Albertazzi, Alberto; Iannone, Anna

not available

2001 - Cytokine and nitric oxide redox regulation in vivo [Abstract in Atti di Convegno]
Tomasi, A; Bellei, E; Bergamini, S; Rota, C; Iannone, A.

Inflammation and oxidative stress, at the molecular level, induce peroxidation and modify redox equlibrium.

2001 - N-Acetylcysteine inhibits in vivo nitric oxide production by inducible nitric oxide synthase [Articolo su rivista]
Bergamini, Stefania; C., Rota; R., Canali; Mg, Staffieri; F., Daneri; Bini, Anna; F., Giovannini; Tomasi, Aldo; Iannone, Anna

non disponibile

2000 - Antioxidative efficacy of parallel and combined supplementation with coenzyme Q10 and d-alpha-trocopherol in mildly hypercholesterolemic subjects: a randomized placebo-controlled clinical study [Articolo su rivista]
J., Kaikkonen; K., Nyyssonen; Tomasi, Aldo; Iannone, Anna; T. P., Tuomainen; E., Porkkala Sarataho; J. T., Salonen

It has been claimed that coenzyme Q10 (Q10) would be an effective plasma antioxidant since it can regenerate plasma vitamin E. To test separate effects and interaction between Q10 and vitamin E in the change of plasma concentrations and in the antioxidative efficiency, we carried out a double-masked, double-blind clinical trial in 40 subjects with mild hypercholesterolemia undergoing statin treatment. Subjects were randomly allocated to parallel groups to receive either Q10 (200 mg daily), d-alpha-tocopherol (700 mg daily), both antioxidants or placebo for 3 months. In addition we investigated the pharmacokinetics of Q10 in a separate one-week substudy. In the group that received both antioxidants, the increase in plasma Q10 concentration was attenuated. Only vitamin E supplementation increased significantly the oxidation resistance of isolated LDL. Simultaneous Q10 supplementation did not increase this antioxidative effect of vitamin E. Q10 supplementation increased and vitamin E decreased significantly the proportion of ubiquinol of total Q10, an indication of plasma redox status in vivo. The supplementations used did not affect the redox status of plasma ascorbic acid. In conclusion, only vitamin E has antioxidative efficiency at high radical flux ex vivo. Attenuation of the proportion of plasma ubiquinol of total Q10 in the vitamin E group may represent in vivo evidence of the Q10-based regeneration of the tocopheryl radicals. In addition, Q10 might attenuate plasma lipid peroxidation in vivo, since there was an increased proportion of plasma ubiquinol of total Q10.

2000 - Ex-vivo detection of free radicals [Capitolo/Saggio]
Tomasi, Aldo; Iannone, Anna; M., Ferrari; V., Quaresima

Ex-vivo detection of free radicals

1999 - 1-Benzopyran-4-one antioxidants as aldose reductase inhibitors [Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; Gamberini, Maria Cristina; Ja, Vinson; P., Bose; Iannone, Anna; M., Staffieri; L., Antolini; A., DEL CORSO; U., Mura; A., Albasini

Starting from the inhibitory activity of the flavonoid Quercetin, a series of 4H-1-benzopyran-4-one derivatives was synthesized and tested for inhibition of aldose reductase, an enzyme involved in the appearance of diabetic complications. Some of the compounds obtained display inhibitory activity similar to that of Sorbinil but are more selective than Quercetin and Sorbinil with respect to the closely related enzyme, aldehyde reductase, and also possess antioxidant activity. Remarkably, these compounds possess higher pK(a) values than carboxylic acids, a characteristic which could make the pharmacokinetics of these compounds very interesting. Molecular modeling investigations on the structures of inhibitors bound at the active site of aldose reductase were performed in order to suggest how these new inhibitors might bind to the enzyme and also to interpret structure-activity relationships.

1999 - Prooxidant activity of ferrioxamine in isolated rat hepatocytes and linoleic acid micelles [Articolo su rivista]
Bergamini, Stefania; C., Rota; M., Staffieri; Tomasi, Aldo; Iannone, Anna

The complex iron-desferrioxamine (ferrioxamine) is considered chemically unreactive, and not able to participate in redox cycle reactions. Desferrioxamine-dependent toxicity is, however, described in both human and animal studies. The aim of this work was to test the possibility that chelated iron, under certain circumstances, could enter redox reactions, giving an explanation of desferrioxamine side effects, Carefully prepared ferrioxamine, to obtain a 1:1 desferrioxamine:iron ratio, was added to isolated rat hepatocytes and to linoleic acid micelles. A strong prooxidant and cytotoxic effect was observed in the cells, also potentiating tert-butyl hydroperoxide-induced lipid peroxidation. In micelles, the prooxidant effect was observed only in the presence of ascorbate, which is oxidized during the process, giving rise to ascorbyl radical. Ferrioxamine, under the experimental conditions used, did not release iron, indicating that the prooxidant effect was due to iron redox cycling. The addition of desferrioxamine prevented both ferrioxamine- and tert-butyl hydroperoxide-induced lipid peroxidation and cytotoxicity. Concurrently, a nitroxide radical was detected, an indication of the radical scavenger activity of the hydroxamic moiety. No radical species was observed when ferrioxamine was added to the same system. The prooxidant effect of ferrioxamine gives a possible explanation of the reported human and animal desferrioxamine toxicity. When, in compartmentalized regions, the ratio of desferrioxamine:metal reaches 1:1, ferrioxamine is formed. In the absence of metal-free desferrioxamine, ferrioxamine can participate in redox cycling reactions, initiating lipid peroxidation and cytotoxicity.

1998 - Antioxidant activity of carotenoids: an electron spin resonance study of beta-carotene and lutein interaction with free radicals generated in a chemical system [Articolo su rivista]
Iannone, Anna; C., Rota; Bergamini, Stefania; Tomasi, Aldo; L. M., Canfield

b-Carotene is thought to be a chainbreaking antioxidant, even though we have no information about the mechanism of its antioxidant activity. Using electron-spin resonance (ESR) spectroscopy coupled to the spin-trapping technique, we have studied the effect of b-carotene and lutein on the radical adducts of the spin-trap PBN (N-t-butyl-a-phenylnitrone) generated by the metal-ion breakdown of different tert-butyl hydroperoxide (tBOOH) concentrations in methylene chloride. The peroxyl radical, along with an oxidation product of PBN (the PBNOx), trapped at room temperature from the breakdown of high concentration of tBOOH (1 M), were quenched by b-carotene or lutein, in competition with the spintrapping agent. However, carotenoids were not able to quench the alkoxyl and methyl radicals generated in the reaction carried out in the presence of low tBOOH concentration (1 mM). The reaction between carotenoids and the peroxyl radical was also carried out in the absence of the spin trap, at 77 K: Under these different experimental conditions, we did not detect any radical species deriving from carotenoids. In the same system, a further evidence of the peroxyl radical quenching by b-carotene and lutein was obtained. The antioxidant activity of vitamin E was also tested, for comparison with the carotenoids. In the presence ofatocopherol, peroxyl and alkoxyl radicals were quenched, and the tocopheroxyl radical was detected. Our data provide the first direct evidence that carotenoids quench peroxyl radicals. Under our experimental conditions, we did not detect any carotenoid radical species that could derive from the interaction with the peroxyl radical. The radical-trapping activity of b-carotene and lutein demonstrated in this chemical reaction contributes to our understanding carotenoid antioxidant action in biological systems.

1998 - Spatial and temporal dynamics of hepatic stellate cell activation during oxidant-stress-induced fibrogenesis [Articolo su rivista]
Montosi, Giuliana; Garuti, Cinzia; Iannone, Anna; Pietrangelo, Antonello

In vitro and in vivo studies indicate that oxidant stress is implicated in Liver fibrogenesis, However, it is still unknown whether, in vivo, oxidant stress directly affects the hepatic cells responsible for fibrogenesis, ie, the hepatic stellate cells (HSCs), This study was aimed at answering this question by assessing the temporal and spatial relationships between oxidant stress and activation of HSCs in an in vivo model of oxidant-stress-associated fibrogenesis, To this purpose, rats were treated with carbon tetrachloride (CCl4) and livers subjected to in situ perfusion with nitroblue tetrazolium, which, in the presence of superoxide ions, is reduced to an insoluble blue-colored formazan derivative and is readily detectable in the tissue by Light microscopy, Moreover, various combinations of in situ hybridization and immunocytochemical analyses were performed. An acute dose of CCl4 caused a transient production of superoxide radicals at 24 hours into pericentral necrotic areas, whereas HSC appearance and expression of collagen mRNA were detectable only at 48 and 72 hours. After chronic CCl4 intoxication, higher levels of oxygen radical production in necrotic areas were detectable along with dramatic and sustained activation of HSCs, However, maximal HSC activation was still delayed as compared with superoxide production. Expression of heme oxygenase, a gene responsive to a variety of oxidant stress mediators, was strongly enhanced by chronic CCl4 administration but remained unchanged in HSCs, both in situ and after isolation of pure HSC fractions from control and CCl4-treated animals. In conclusion, during postnecrotic fibrogenesis, oxidant stress anticipates HSC activation. HSCs do not directly face an oxidant stress while engaged in active fibrogenesis.

1997 - Application of electron spin resonance-spin trapping (EPR-ST) to the demonstration of biologically relevant free radicals [Capitolo/Saggio]
A., Kozlov; Iannone, Anna; I. ZINI AND A., Tomasi

Application of electron spin resonance-spin trapping (EPR-ST) to the demonstration of biologically relevant free radicals

1997 - The rat liver during carbontetrachloride and 1,2-dibromoethane intoxication: a study on biochemical injury and energy state alteration [Articolo su rivista]
Tomasi, A.; Bini, A.; Ghelli, S.; Iannone, Anna; Meletti, E.; Vannini, U. MUSCATELLO AND V.

The rat liver during carbontetrachloride and 1,2-dibromoethane intoxication: a study on biochemical injury and energy state alteration

1996 - 'Free' iron, as detected by electron paramagnetic resonance spectroscopy, increases unequally in different tissues during dietary iron overload in the rat [Articolo su rivista]
A. V., Kozlov; Bini, Anna; D., Gallesi; F., Giovannini; Iannone, Anna; A., Masini; E., Meletti; Tomasi, Aldo

'Free' iron concentration, as determined by electron paramagnetic resonance (EPR) spectroscopy, and lipid peroxidation (LPO), as determined by thiobarbituric acid test, were assessed in the lung, heart, liver, spleen, brain and kidney of rats subjected to experimental iron overload, Two tests, Desferal- and NO-available iron, were used to measure 'free' iron and gave comparable results, The most pronounced accumulation of 'free' iron was observed in liver, kidney and spleen, Differences between control and iron loaded animals increased during the initial 90 days of treatment, Between 90 and 180 days 'free' iron concentration reached a steady state level, or even decreased, as in the case of liver, Lipid peroxidation level, measured in the organs of both treated and matched controls, did not give any significant difference during the initial 90 days of treatment, A significant augmentation was observed in liver, kidney, spleen and heart at 180 days, The results of the present research show that, under conditions of moderate siderosis, the occurrence of LPO is partially related to the level of 'free' iron.

1996 - Electron paramagnetic resonance characterization of rat neuronal nitric oxide production ex vivo [Capitolo/Saggio]
Av, Kozlov; Bini, Anna; Iannone, Anna; Zini, Isabella; Tomasi, Aldo


1996 - Generation of N-tert-butyl-alpha-phenylnitrone radical adducts in iron breakdown of tert-butyl-hydroperoxide [Articolo su rivista]
Iannone, Anna; Tomasi, Aldo; Canfield, Lm

ESR spectroscopy coupled to the spin trapping technique was used to evaluate the generation of radical species arising from the ferrous ion induced decomposition of tert-butyl hydroperoxide ('BuOOH) in methylene chloride. We report here that N-tert-butyl-alpha-phenylnitrone (PEN) can trap peroxyl radicals generated in the ferrous ion induced breakdown of high concentration of 'BuOOH (1M) at room temperature, the radical adduct being stable under the light. The peroxyl radical formation was demonstrated by direct ESR measurements at 77K. In contrast, alkoxyl and methyl radicals were trapped only in the presence of low hydroperoxide concentration (1mM). In order to measure the hyperfine splitting constants (hfsc) of the PEN-methyl adduct spectra were obtained in the presence of diphenylamine (DPA) or 2,6-di-tert-butyl-4-methylphenol (BHT), which quenched the alkoxyl radical. For this latter radical, the hfsc were calculated by computer simulation. A mechanism for a direct interaction between DPA and the alkoxyl radical is presented. DPA quenched the peroxyl radical in the reaction of high hydroperoxide concentrations, with the concomitant generation of a DPA nitrogen-based radical.

1995 - Actvation of alkylhydrazines to free radical intermediates by ethanol-inducible cytochrome P4502E1 (CYP2E1) [Articolo su rivista]
Albano, E; Comoglio, A; Clot, P; Iannone, Anna; Tomasi, Aldo; INGELMAN SUNDBERG, I.

Electron spin resonance (EPR) spectroscopy analysis using the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) was used to measure the formation of free radical intermediates during NADPH-dependent oxidation of 1-methyl-, 1-ethyl-, and 1-isopropylhydrazine in rat liver microsomes and in reconstituted enzyme systems. The experiments in microsomes revealed that the specific activation of the hydrazines, as measured by the EPR signal intensities, was about two-fold higher, when expressed per nmol of P-450, in microsomes from rats treated with ethanol (EtOH) as compared to membranes isolated from either phenobarbital (PB)-, beta-naphthoflavone (beta-NF)-treated or control rats. Furthermore, kinetic experiments revealed that EtOH-microsomes had an apparent affinity for 1-ethylhydrazine about one order of magnitude higher than PB-microsomes. In reconstituted vesicular systems composed of phospholipids, NADPH cytochrome P-450 reductase and P-450, the intensities of EPR signals produced by the formation of the methyl-, ethyl- and isopropyl-free radicals, were 3- to 5-fold more intense in membrane vesicles containing ethanol-inducible CYP2E1 than phenobarbital-inducible CYP2B1. By contrast, CYP1A2, CYP2B4 and CYP2C4 were inefficient catalysts of radical formation. Desferrioxamine, catalase and superoxide dismutase did not influence the extent of ethyl radicals formed in EtOH-microsomes, indicating that hydroxyl radicals are not involved in the CYP2E1-dependent activation of 1-ethylhydrazine. Addition of cytochrome b5, an efficient donor of the second electron to P-450 and hence an inhibitor of the formation of the oxy-cytochrome P-450 complex, increased to be consistent with the results, did not influence the amount of ethyl radicals trapped. In liver microsomes from untreated rats selective substrates of CYP2E1, such as diethyl-dithiocarbamate and p-nitrophenol, as well as anti-CYP2E1-IgG, inhibited the free radical formation from 1-ethylhydrazine by about 60%. The anti-CYP2E1 IgG used significantly inhibited ethyl radical production also in human liver microsomes incubated with 1-ethylhydrazine and 4-POBN. Taken together, these results indicate that CYP2E1, as compared to other rat liver cytochromes P-450, is an efficient catalyst of transformation of alkylhydrazines to free radical intermediates, a finding that might be of importance in the development of the toxicity of these compounds.

1995 - Effect of acute exercise and L-carnitine administration on rat liver mitochondria [Articolo su rivista]
V., Bobyleva Guarriero; Iannone, Anna; F., Di Lisa; R., Capone; N., Siliprandi

The present study investigated the effect of acute physical exercise and L-carnitine treatment on the respiratory functions of rat liver mitochondria. This work clearly demonstrates that physical exercise induces a significant increase in state 3 respiration, which is further enhanced by the concomitant L-carnitine administration.

1994 - Hydroperoxide metabolism in cultured normal human Keratinocytes a spin trapping study [Capitolo/Saggio]
Giannetti, Alberto; Marconi, Alessandra; G., Zambruno; Iannone, Anna; Tomasi, Aldo


1993 - Free-Radical Production During Metabolism Of Organic Hydroperoxides By Normal Human Keratinocytes [Articolo su rivista]
Iannone, Anna; Marconi, Alessandra; G., Zambruno; Giannetti, Alberto; V., Vannini; Tomasi, Aldo

Evidence of a relationship between tumor production induced by various organic (hydro)peroxides and free radical formation has been shown in cultured murine keratinocytes and human skin-tumor cell line. In the present study the bioactivation of cumene hydroperoxide, t-butyl-hydroperoxide, and benzoyl peroxide via one-electron oxidation or reduction was compared in freshly isolated and in cultured normal human keratinocytes. The formation of methyl free radicals during the metabolism of cumene and t-butyl-hydroperoxide was shown by the electron spin resonance-spin trapping technique. Radical formation increased under hypoxic conditions. An intracellular activation site was demonstrated by the use of two spin-trapping agents, the hydrophilic, membrane-impermeable, 3,5-dibromo-4-nitrosobenzenesulfonic acid and the lipophilic, membrane-permeable alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone. At 30 min incubation and 25 mM concentration, hydroperoxides exhibited cytotoxicity, as indicated by trypan blue exclusion and lactate dehydrogenase release assay; free radicals were concurrently trapped. Hydroperoxides at a lower concentration (1 mM) did not significantly affect cell viability. However, free radical production was still detected using a membrane-permeable spin trap. The incubation of keratinocytes with benzoyl peroxide did not show any peroxide-dependent radical adduct. No significant differences in bioactivation capability were demonstrated between freshly isolated and cultured human keratinocytes. The results indicate that cultured human keratinocytes can be used as a model system for the study of the metabolic activation to free radical intermediates of toxic and carcinogenic compounds in the epidermis.

1993 - Nitroxides as metabolic and EPR imaging probes in biological model systems [Articolo su rivista]
Iannone, Anna; Tomasi, Aldo; Quaresima, V; Ferrari, M.

Nitroxides are unusually stable free radicals and sensitive ''reporters'' of the chemico-physical environment which surround them. The term ''reporter group'' was used to indicate this property, later the term spin label or spin probe was preferred. The facile synthesis and the multiplicity of compounds which have been coupled to the nitroxide moiety has allowed the development of a number of applications. Nitroxides have been used to study molecular mobility of proteins and lipids in membranes, enzyme active sites, DNA synthesis, and measure of electrical potential, pH, temperature as well as oxygen gradients across membranes; more recently, as contrast agents in magnetic resonance imaging and electron paramagnetic resonance imaging (EPRI). Aim of this work is to review and comment on the use of various nitroxides as metabolic probes, and in EPRI in studies of biomedical interest.

1993 - Possible role of free radical intermediates in hepatotoxicity of hydrazines derivatives [Articolo su rivista]
Albano, E; Goriagatti, L; Clot, P; Iannone, Anna; Tomasi, Aldo

Hydrazine derivatives constitute a wide group of compounds and have found application in industry, agriculture, and (as therapeutical agents) medicine. In spite of their widely spread use, several hydrazine derivatives are known to exert hepatotoxic effects and are carcinogenic. Free radical species are produced during the hepatic biotransformation of alkylhydrazines by both rat and humans liver microsomes. Cytochrome P-450 dependent monoxygenase system is responsible for the production of these reactive species and specific cytochrome P-450 isoenzymes appear to catalyze the formation of free radical intermediates. Free radicals generated during the metabolism of alkylhydrazines are capable of inducing oxidative stress in isolated hepatocytes and might contribute to the development of cell injury.

1993 - t-Butyl-hydroperoxide bioactivation to methyl radical in rat liver mitochondria and submitochondrial particles [Articolo su rivista]
Iannone, Anna; Bini, A; JIN Y., G; Tomasi, Aldo; Vannini, V.

Electron paramagnetic resonance spectroscopy (EPR) coupled to the spin trapping technique was used to detect carbon-centered radicals in rat liver mitochondria and submitochondrial particles exposed to t-butyl-hydroperoxide (TBH), using the spin trapping agent 3,5-dibromo-4-nitroso-benzenesulfonic acid (DBNBS). The signal recorded was unambiguously assigned to the methyl radical adduct. DBNBS was added to isolated rat liver mitochondria energized with succinate, and the methyl radical adduct was observed. The addition of NADH, NADPH, inhibitors of the respiratory chain, and of monoaminoxidase (MAO) inhibitors did not cause any relevant modification in the yield of radical adduct formation. Boiling and the addition of a non-ionic detergent inhibited the formation of the radical adduct, while experiments carried out under hypoxic conditions generated a significant increase in methyl radical formation. Further experiments were carried out on sub-mitochondrial particles (SMP) giving rise to, basically, the same results. From the above results, we are proposing that haem prosthetic groups are the likely source of TBH bioactivation in mitochondria.

Iannone, Anna; Federico, Massimo; Tomasi, Aldo; Magin, Rl; Casasco, A; Calligaro, A; Vannini, V.

RATIONALE AND OBJECTIVES. The compound studied in this article is a superparamagnetic macromolecular complex of magnetite cores coated with hydrophilic dextran, which is under active investigation as a contrast agent for magnetic resonance imaging (MRI) in liver and spleen. The biodistribution of paramagnetic compounds is problematic and is usually studied by histochemical reactions or by radiolabeling the compound under study. The purpose of this article is to show how electron spin resonance (ESR) spectroscopy detects dextran magnetite (DM) particles in tissues. METHODS. DM injected intravenously in the experimental animal was detected in some reticulo-endothelial organs by ESR. The spectroscopic study was validated using electron microscopy and electron-probe microanalysis. RESULTS. DM exhibits an ESR spectrum; ESR delineated the distribution of DM distribution in liver, spleen, bone marrow, and blood as a function of time. The blood clearance was biphasic, dependent on the size of particles. CONCLUSIONS. ESR spectroscopy is a highly sensitive and reproducible method of studying DM distribution.

1992 - In vitro and in vivo evidence for the formation of methyl radical from procarbazine: a spin trapping study [Articolo su rivista]
GORIA GATTI, L; Iannone, Anna; Tomasi, Aldo; Poli, G; Albano, E.

Electron spin resonance (ESR) analysis combined with the use of 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) and dibromonitroso benzenesulfonic acid (DBNBS) as spin-trapping agents was used to characterize free radical generation during the metabolism of the anticancer agent procarbazine [N-isopropyl-a-(2-methylhydrazino)-p-toluamide hydrochloride]. The formation of free radical species, identified as methyl radicals, was observed during oxidation of procarbazine in rat liver microsomes and isolated hepatocytes in vitro, as well as in several organs following administration of the drug in vivo. A cytochrome P450-mediated reaction, involving P450IA and IIB isoenzymes, was responsible for the activation process. The metabolic pathway leading to free radical formation was characterized using various procarbazine metabolites and revealed strict analogies with previously published data on methane production from procarbazine. These results supported the identification of the trapped species as methyl free radical and suggested that C-oxidation of azoprocarbazine is the main source of radical intermediates derived from this anticancer drug.

1991 - Blood clearance of dextran magnetite particles by a non invasive in vivo ESR method [Articolo su rivista]
Iannone, Anna; Magin, R. L.; Walczac, T.; Federico, Massimo; Swartz, H. M.; Tomasi, Aldo; Vannini, V.

Dextran magnetite (DM) is a potential MR contrast agent with superparamagnetic properties. Its fast clearance from the blood and selective uptake by tissue macrophages provide advantages for imaging tumors in the liver and spleen. DM consists of a suspension of solid particles with a wide distribution of sizes. In this study we have used ESR spectroscopy to determine the blood clearance of DM injected iv in mice. The spectra are obtained on living animals by inserting the tail of a mice into the waveguide cavity of the ESR spectrometer and recording the ESR spectrum continuously. This procedure allows the direct measurement of the plasma clearance of DM from individual animals, without blood sampling. We applied this method to study the clearance of suspensions of DM particles with different average sizes.

Iannone, Anna; Tomasi, Aldo

The concept of using a probe, sensitive to the environment, termed >>reporter group<<, arose in the early sixties; readily, nitroxides became the natural candidate for a >>reporter group<<. The term >>reporter group<< was later dismissed and the term spin label or spin probe preferred. The facile synthesis and the multiplicity of compounds which have been coupled to the nitroxide moiety has allowed the development of a number of applications including molecular mobility of proteins and lipids in membranes, study of the enzyme active site and DNA synthesis, measurement of electrical potential, measurement of pH, temperature and oxygen gradients across membranes and, more recently, as contrast agents in magnetic resonance imaging and electron paramagnetic resonance imaging. The overview, by no means comprehensive of the complete literature, aims to give an insight view of nitroxide applications as metabolic probes in the field of biomedical studies reporting methodologies and trying to give a critical view of the procedures and methodologies used.

1990 - Free radical detection during xenobiotics metabolism by human keratinocytes [Capitolo/Saggio]
Giannetti, Alberto; Marconi, Alessandra; M. L., Santantonio; G., Zambruno; Iannone, Anna; Tomasi, Aldo


Iannone, Anna; Tomasi, Aldo; Vannini, V; Swartz, Hm

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding NADPH, induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADH or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.

Iannone, Anna; Tomasi, Aldo; Vannini, V; Swartz, Hm

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of five nitroxides in cytosol derived from rat hepatocytes. The nitroxides were chosen to provide information on the effects of the type of charge and the ring on which the nitroxyl group is located. The rates of reduction were fastest for a six-membered positively charged nitroxide ('CAT-1') and slowest for an anionic five-membered ring nitroxide ('PCA'). Changing levels of glutathione, sulphydryl groups in general, NADPH or NADH had little or no effect on the rates of reduction, while the addition of ascorbate oxidase essentially abolished reduction of the nitroxides. The products of reduction by the cytosol were the corresponding hydroxylamines. The overall rates of reduction of neutral or anionic nitroxides were much slower than those observed with intact cells. We conclude that the primary source of metabolism of nitroxides by cytosol is reduction by ascorbate and that under most conditions reduction of nitroxides in the cytosol is not a major factor in the metabolism of nitroxides by cells.

1989 - Ascorbyl radical is detected in rat isolated hepatocytes suspensions undergoing oxidative stress: an early index of oxidative damage in cells [Relazione in Atti di Convegno]
Tomasi, Aldo; E., Albano; A., Bini; Iannone, Anna; V., Vannini

In this article we have studied the formation of free radical intermediates induced by paraquat, whose toxicity is well known, and by the anti-tumour agent adriamycin in isolated rat hepatocytes comparing the results to those obtained using an iron chelate, such as ADP-iron, a strong inducer of lipid peroxidation. Both paraquat and adriamycin give rise to toxic reactive species of oxygen, our goal being that of inducing an oxidative stress in the model system used.

1989 - Bone marrow uptake of liposome-entrapped spin label after liver blockade with empty liposomes [Articolo su rivista]
Federico, Massimo; Iannone, Anna; Chan, H. C.; Magin, R. L.

Using an ESR spectrometer, we studied the time course of the uptake of the liposome-entrapped spin label 2,2,6,6-tetramethylpiperidine-N-oxyl-4-trimethylammonium in liver, spleen, and bone marrow following reticuloendothelial liver blockade. Our results show that suppression of the phagocytic activity of the liver increases the delivery of liposomes to the spleen and bone marrow without substantially altering uptake by the liver.

1989 - Free radical activation of monomethyl and dimethyl hydrazines in isolated hepatocytes and liver microsomes [Articolo su rivista]
E., Albano; Tomasi, Aldo; L., Goria Gatti; Iannone, Anna

Isolated hepatocytes and liver microsomes incubated with monomethyl-1,1 dimethyl- and 1,2 dimethyl-hydrazines produced free radical intermediates which were detected by ESR spectroscopy by using 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) as spin trapping agent. The spectral features of the spin adducts derived from all three hydrazine derivatives corresponded to the values reported for the methyl free radical adduct of 4-POBN. In the microsomal preparations inhibitors of the mixed function oxidase system and the destruction of cytochrome P450 by pretreating the rats with CoCl2 all decreased the free radical formation. Methimazole, an inhibitor of FAD-containing monoxygenase system, similarly decreased the activation of 1,1 dimethyl-hydrazine, but not that of monomethyl- and 1,2 dimethyl-hydrazines. The addition to liver microsomes of physiological concentrations of glutathione (GSH) lowered by approx. 80% the intensities of the ESR signals. Consistently, incubation of isolated hepatocytes with methyl-hydrazines decreased the intracellular GSH content, suggesting that GSH can effectively scavenge the methyl free radicals. The results obtained suggest that methyl free radicals could be the alkylating species responsible for the toxic and/or carcinogenic effect of methyl-hydrazines.

1989 - Free radicals and lipid peroxidation in liver of rats kept on a diet devoid of choline [Articolo su rivista]
S., Banni; F. P., Corongiu; M. A., Dessì; Iannone, Anna; B., Lombardi; Tomasi, Aldo; V., Vannini

Rodents kept on a choline devoid (CD) diet up to 14 months develop hepatic lesions progressing through two broad stages. The first is characterized by severe steatosis and increase in cell turnover, the second by a gradual clearance of the deposited fat and fibrosis. Hepatocellular carcinomas eventually arise in rats fed for over 12 months, even though the animals aer not exposed to chemical carcinogens. It has been suggested that the diet may trigger generated thereby may be responsible for initiation of liver cancer and promotion. The radicals would lead to DNA damage, and the altered DNA in a proliferating liver would result in initiation of the carcinogenic process. In this communication we present evidence that the diet used in the above studies contained stable fatty acid isomers with conjugated dienes, which are absorbed and deposited in rat liver. This finding cast doubts on whether a CD diet does indeed cause a peroxidation of cellular membrane lipids. Electron spin resonance (ESR) spectroscopy was also used to investigate whether any abnormal pattern of free radicals exists in the liver of rats fed a CD diet. No significant differences were noted in ESR spectra of either transition metal-centered signals, or organic free radicals.

1989 - Metabolism in rat liver microsomes of the nitroxide spin probe Tempol [Articolo su rivista]
Iannone, Anna; A., Bini; H. M., Swartz; Tomasi, Aldo; V., Vannini

Paramagnetic nitroxide spin labels have been extensively used to probe various biophysical and biochemical properties of the cellular environment. Recently nitroxides have been proposed as contrast enhancing agents in proton magnetic resonance imaging and contrast enhancement has been demonstrated in animal studies. Nitroxides, possessing a stable unpaired electron, increases the relaxation rates of protons, providing an enhancement of contrast. Nitroxides are metabolized intracellularly principally via reversible reduction to hydroxylamines. Rates of reduction depend on the physical characteristics of the nitroxides, in general 5-membered pyrrolidine ring are reduced more slowly than those with a 6-membered piperidine ring. Oxidation back to the nitroxide is relevant for lipid soluble hydroxylamines, while is low for water soluble ones. It is known that nitroxides are metabolized by subcellular fractions (cytosol, mitochondria, microsomes), though the enzymatic and non-enzymatic systems involved are poorly characterized. In the present study, the first of the necessary steps toward a systematic study of the metabolism of nitroxides by subcellular organelles, we have chosen to study the metabolism of 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl in isolated rat liver microsomes. Microsomes were able to reduce Tempol slowly without any substrate addition; when NADPH was added, the reduction rate substantially increased. In phenobarbitone induced rats the reduction rate was significantly higher than in not-induced microsomes. NADPH-dependent reduction rate was inhibited by thallium chloride (an inhibitor of the flavin-centered cytochrome P-450 reductase), superoxide dismutase, and by N-ethylmaleimide; menadione increased it. The Tempol reduction rate was not significantly affected by various cytochrome P-450 inhibitors with the sole exception of metyrapone. A solution containing purified cytochrome P-450 reductase and NADPH readily reduced Tempol. Microsomes fortified with NADPH were able to reduce Tempol at an appreciable rate. In order to distinguish between reduction of nitroxides to hydroxylamine or destruction of nitroxides following nitroxide reduction, microsomal suspensions were treated with a mild oxidant (ferricyanide 0.5-10 mM). The recovery varied from 40 to 60%, indicating a process of probe destruction leading to as yet unknown metabolites. The present study clearly indicates that, in this model system, cytochrome c (P-450) reductase and not cytochrome P-450 is responsible for the observed Tempol metabolism; along with hydroxylamine formation, other Tempol derived metabolites are formed during the process.

1989 - Metabolism of aqueous soluble nitroxides in hepatocytes: effects of cell integrity, oxygen, and structure of nitroxides [Articolo su rivista]
Iannone, Anna; H., Hu; Tomasi, Aldo; V., Vannini; H. M., Swartz

The optimum use of nitroxides in viable biological systems, including live animals, requires knowledge of the metabolism of nitroxides by major organ systems, especially the liver. We report here details of the metabolism of several prototypic aqueous soluble nitroxides in suspensions of freshly isolated hepatocytes. The general patterns of metabolism were similar to those observed in other types of cells (previous studies have been done principally in cells from tissue culture, such as CHO cells) including the primary initial reaction being reduction to the hydroxylamine, an increased rate of metabolism of some nitroxides in hypoxic cells, faster rates of reduction of nitroxides on six-membered piperidine rings compared to five-membered pyrrolidine rings, and most metabolism being intracellular. Metabolism in hepatocytes differed from other cell lines in having (1) significant reduction in the extracellular medium due to ascorbate that was released from damaged hepatocytes; (2) decreased rates of metabolism in freeze-thawed cells due to damage to subcellular organelles. These results provide much of the data needed to understand the role of the liver in the metabolism of nitroxides by intact animals and explain some previously puzzling results which indicated an apparent unusually high rate of metabolism of a charged nitroxide (Cat1) by hepatocytes. Our results also indicate that the use of freshly isolated cells or tissue homogenates may introduce experimental artifacts in the study of the metabolism of nitroxides

1989 - NMR study of water exchange across the hepatocyte membrane [Articolo su rivista]
G., Bacic; J. C., Alameda; Iannone, Anna; R. L., Magin; H. M., Swartz

An understanding of the cellular permeability for water is needed to evaluate MR images of complex tissues, such as liver, and to interpret the effects of contrast agents. To obtain data essential for such an understanding we measured water exchange across the isolated rodent hepatocyte membrane by proton NMR relaxation with dextranmagnetite as a relaxation agent. The results are treated as water exchange in a two-compartment system, and possible reasons for deviations from that behavior are analyzed. The mean residence time of intracellular water was approximately 40 ms at 37 degrees C. We found the lower limit for the diffusional permeability of the hepatocyte membrane to be 8 x 10(-3) cm s-1. These results, combined with consideration of hepatic anatomy indicate that the failure to observe effects on the T1 of liver from particulate contrast agents such as magnetite, Gd-starch, and liposome encapsulated Mn2+ is due to the localization of these agents in the Kupffer cells. Also, the nonexponential T1 decay observed in normal liver is unlikely to be due to slow exchange of water between compartments.

1988 - Effects of single or multiple doses of L-carnitine on liver energetic metabolism of rats forced to run [Articolo su rivista]
Bobyleva Guarriero, V; Iannone, Anna; Bellei, Monica; Muscatello, U.

The effects of treatment with a single dose of L-carnitine and of daily injection of carnitine for one month on liver energetic metabolism in both sedentary and acutely exercised rats were compared. The results show that the treatment with a single dose of carnitine enhances the aerobic metabolism of liver mitochondria in both resting and in running rats. On the contrary, the daily injection of carnitine for one month does not cause any significant effect on the aerobic metabolism of liver. Treatment with single or multiple doses of carnitine does not modify the serum concentration of cholesterol and of triglycerides in comparison with respective controls.

1987 - Free radical metabolism of carbon tetrachloride in rat liver mitochondria. A study of the mechanism of activation [Articolo su rivista]
TOMASI, Aldo; E., Albano; S., Banni; B., Botti; F. P., Corongiu; M. A., Dessì; IANNONE, Anna; VANNINI, Vanio; M. U., Dianzani

Alterations in liver mitochondria as consequence of rat poisoning with carbon tetrachloride (CCl4) have been reported over many years, but the mechanisms responsible for causing such damage are still largely unknown. Isolated rat liver mitochondria incubated under hypoxic conditions with succinate and ADP were found able to activate CCl4 to a free-radical species identified as trichloromethyl free radical (CCl3) by e.s.r. spectroscopy coupled with the spin-trapping technique. The incubation of mitochondria in air decreased free-radical production, indicating that a reductive reaction was involved in the activation of CCl4. However, in contrast with liver microsomes (microsomal fractions), mitochondria did not require the presence of NADPH, and the process was not significantly influenced by inhibitors of cytochrome P-450. The addition of inhibitors of the respiratory chain such as antimycin A and KCN decreased free-radical formation by only 30%, whereas rotenone displayed a greater effect (approx. 84% inhibition), but only when preincubated for 15 min with mitochondria not supplemented with succinate. These findings suggest that the mitochondrial electron-transport chain is responsible for the activation of CCl4. A conjugated-diene band was observed in the lipids extracted from mitochondria incubated with CCl4 under anaerobic conditions, indicating that stimulation of lipid peroxidation was occurring as a result of the formation of free-radical species.

1985 - Ameliorating effect of carnitine on liver mitochondria functions in ammonium intoxicated rats [Articolo su rivista]
V., Bobyleva Guarriero; F., Di Lisa; Iannone, Anna; N., Siliprandi

We have studied some functions of mitochondria isolated from livers of rats previously treated with ammonium acetate or with this salt plus carnitine. The results reported here show that carnitine administration exerts a protective action against functional damage caused by ammonium acetate in the liver mitochondria of treated animals